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Sample records for gene oct4b mrna

  1. Altered expression of apoptotic genes in response to OCT4B1 suppression in human tumor cell lines.

    PubMed

    Mirzaei, Mohammad Reza; Najafi, Ali; Arababadi, Mohammad Kazemi; Asadi, Malek Hosein; Mowla, Seyed Javad

    2014-10-01

    OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1. PMID:25008565

  2. Down-regulation of HSP40 gene family following OCT4B1 suppression in human tumor cell lines

    PubMed Central

    Mirzaei, Mohammad Reza; Asadi, MalekHosein; Mowla, Seyed Javad; Hassanshahi, Gholamhossin; Ahmadi, Zahra

    2016-01-01

    Objective(s): The OCT4B1, as one of OCT4 variants, is expressed in cancer cell lines and tissues more than other variants and plays an important role in apoptosis and stress (heat shock protein) pathways. The present study was designed to determine the effects of OCT4B1 silencing on expressional profile of HSP40 gene family expression in three different human tumor cell lines. Materials and Methods: The OCT4B1 expression was suppressed by specific siRNA transfection in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines employing Lipofectamine reagent. Real-time PCR array technique was employed for RNA qualification. The fold changes were calculated using RT2 Profiler PCR array data analysis software version 3.5. Results: Our results indicated that fifteen genes (from 36 studied genes) were down-regulated and two genes (DNAJC11 and DNAJC5B) were up-regulated in all three studied tumor cell lines by approximately more than two folds. The result of other studied genes (19 genes) showed different expressional pattern (up or down-expression) based on tumor cell lines. Conclusion: According to the findings of the present study, we may suggest that there is a direct correlation between OCT4B1 expression in tumor cell lines (and tissues) and HSP40 family gene expressions to escape from apoptosis and cancer expansion. PMID:27081464

  3. Altered Expression of High Molecular Weight Heat Shock Proteins after OCT4B1 Suppression in Human Tumor Cell Lines

    PubMed Central

    Mirzaei, Mohammad Reza; Kazemi Arababadi, Mohammad; Asadi, Malek Hossein; Mowla, Seyed Javad

    2016-01-01

    Objective OCT4B1, a novel variant of OCT4, is expressed in cancer cell lines and tis- sues. Based on our previous reports, OCT4B1 appears to have a crucial role in regulating apoptosis as well as stress response [heat shock proteins (HSPs)] pathways. The aim of the present study was to determine the effects of OCT4B1 silencing on the expression of high molecular weight HSPs in three different human tumor cell lines. Materials and Methods In this experimental study, OCT4B1 expression was suppressed in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines using RNAi strategy. Real-time polymerase chain reaction (PCR) array was em- ployed for expression level analysis and the fold changes were calculated using RT2 Pro- filer PCR array data analysis software version 3.5. Results Our data revealed up-regulation of HSPD1 (from HSP60 family) as well as HSPA14, HSPA1L, HSPA4, HSPA5 and HSPA8 (from HSP70 family) following OCT4B1 knock-down in all three cell lines. In contrast, the expression of HSP90AA1 and HSP90AB1 (from HSP90 family) as well as HSPA1B and HSPA6 (from HSP70 family) was down-regulated under similar conditions. Other stress-related genes showed varying ex- pression pattern in the examined tumor cell lines. Conclusion Our data suggest a direct or indirect correlation between the expression of OCT4B1 and HSP90 gene family. However, OCT4B1 expression was not strongly corre- lated with the expression of HSP70 and HSP60 gene families. PMID:26862520

  4. Oct4B, CD90, and CD73 are upregulated in bladder tissue following electro-resection of the bladder

    PubMed Central

    Takeuchi, Takumi; Tonooka, Akiko; Okuno, Yumiko; Hattori-Kato, Mami; Mikami, Koji

    2016-01-01

    Aim: We tested the hypothesis that stimulation by electro-resection of bladder tissue induces stem cells in the tissue repair process. Materials & Methods: After primary transurethral resection of a bladder tumor and surrounding tissue (TUR-Bt), second TUR-Bt was performed. Tissues excised by second TUR-Bt were immunohistochemically stained for Oct4, a marker of pluripotency, and for CD90 and CD73, markers of mesenchymal stromal cells, when no bladder tumor cells remained. Results and Conclusions: Oct4B protein was sporadically stained in the cytoplasm of interstitial cells in four out of eight cases. CD90 and CD73 are upregulated in interstitial and vascular endothelial cells without CD45 expression. Mesenchymal stromal cells, but not pluripotent stem cells, may be mainly involved in bladder tissue repair. PMID:27397997

  5. Gene regulation by structured mRNA elements.

    PubMed

    Wachter, Andreas

    2014-05-01

    The precise temporal and spatial coordination of gene activity, based on the integration of internal and external signals, is crucial for the accurate functioning of all biological processes. Although the basic principles of gene expression were established some 60 years ago, recent research has revealed a surprising complexity in the control of gene activity. Many of these gene regulatory mechanisms occur at the level of the mRNA, including sophisticated gene control tasks mediated by structured mRNA elements. We now know that mRNA folds can serve as highly specific receptors for various types of molecules, as exemplified by metabolite-binding riboswitches, and interfere with pro- and eukaryotic gene expression at the level of transcription, translation, and RNA processing. Gene regulation by structured mRNA elements comprises versatile strategies including self-cleaving ribozymes, RNA-folding-mediated occlusion or presentation of cis-regulatory sequences, and sequestration of trans-acting factors including other RNAs and proteins. PMID:24780087

  6. mRNA modifications: Dynamic regulators of gene expression?

    PubMed Central

    Hoernes, Thomas Philipp; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-01

    ABSTRACT The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methyladenosine (m1A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression. PMID:27351916

  7. mRNA Composition and Control of Bacterial Gene Expression

    PubMed Central

    Liang, S.-T.; Xu, Y.-C.; Dennis, P.; Bremer, H.

    2000-01-01

    The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of β-galactosidase expressed from three different promoter-lacZ fusions (Pspc-lacZ, PRNAI-lacZ, and PRNAII-lacZ) was measured (i) in a relA+ background during exponential growth at different rates and (ii) in relA+ and ΔrelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp. PMID:10809680

  8. GeneTack database: genes with frameshifts in prokaryotic genomes and eukaryotic mRNA sequences.

    PubMed

    Antonov, Ivan; Baranov, Pavel; Borodovsky, Mark

    2013-01-01

    Database annotations of prokaryotic genomes and eukaryotic mRNA sequences pay relatively low attention to frame transitions that disrupt protein-coding genes. Frame transitions (frameshifts) could be caused by sequencing errors or indel mutations inside protein-coding regions. Other observed frameshifts are related to recoding events (that evolved to control expression of some genes). Earlier, we have developed an algorithm and software program GeneTack for ab initio frameshift finding in intronless genes. Here, we describe a database (freely available at http://topaz.gatech.edu/GeneTack/db.html) containing genes with frameshifts (fs-genes) predicted by GeneTack. The database includes 206 991 fs-genes from 1106 complete prokaryotic genomes and 45 295 frameshifts predicted in mRNA sequences from 100 eukaryotic genomes. The whole set of fs-genes was grouped into clusters based on sequence similarity between fs-proteins (conceptually translated fs-genes), conservation of the frameshift position and frameshift direction (-1, +1). The fs-genes can be retrieved by similarity search to a given query sequence via a web interface, by fs-gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, etc. The largest clusters contain fs-genes with programed frameshifts (related to recoding events). PMID:23161689

  9. Stability regulation of mRNA and the control of gene expression.

    PubMed

    Cheadle, Chris; Fan, Jinshui; Cho-Chung, Yoon S; Werner, Thomas; Ray, Jill; Do, Lana; Gorospe, Myriam; Becker, Kevin G

    2005-11-01

    Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. Standard techniques measure changes in total cellular poly(A) mRNA levels. The assumption that changes in gene expression as measured by these techniques are directly and well correlated with changes in rates of new gene synthesis form the basis of attempts to connect coordinated changes in gene expression with shared transcription regulatory elements. Yet systematic attempts at this approach remain difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Recent technical advances have led to the successful scale-up and application of nuclear run-on procedures directly to microarrays. This development has allowed a gene-by-gene comparison between new gene synthesis in the nucleus and measured changes in total cellular polyA mRNA. Results from these studies have begun to challenge the strict interpretation of changes in gene expression measured by conventional microarrays as being closely correlated with changes in mRNA transcription rate, but rather they tend to support the significant expansion of the role played by changes in mRNA stability regulation to standard analyses of gene expression. Gene expression profiles obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in total cellular polyA mRNA in this system. Stability regulation was inferred by the absence of corresponding regulation of nuclear gene transcription activity for groups of genes strongly regulated at the whole cell level and which were also resistant to inhibition by Actinomycin

  10. Circadian oscillations in period gene mRNA levels are transcriptionally regulated.

    PubMed Central

    Hardin, P E; Hall, J C; Rosbash, M

    1992-01-01

    The period (per) gene is involved in regulating circadian rhythms in Drosophila melanogaster. The per gene is expressed in a circadian manner, where fluctuations in per mRNA abundance are influenced by its own translation product, which also cycles in abundance. Since per gene expression is necessary for circadian rhythmicity, we sought to determine how certain features of this feedback loop operate. The results of this study reveal that fluctuations in per mRNA are primarily controlled by fluctuations in per gene transcription, that per mRNA has a relatively short half-life, and that sequences sufficient to drive per mRNA cycling are present in 1.3 kilobases of 5' flanking sequences. These and other results indicate that the per feedback loop has all of the basic properties necessary to be a component of a circadian oscillator. Images PMID:1465387

  11. Nonsense mutations in the human. beta. -globin gene affect mRNA metabolism

    SciTech Connect

    Baserga, S.J.; Benz, E.J. Jr. )

    1988-04-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.

  12. Genetic organization and mRNA expression of enolase genes of Candida albicans.

    PubMed

    Postlethwait, P; Sundstrom, P

    1995-04-01

    In previous work, we cloned a Candida albicans cDNA for the glycolytic enzyme enolase and found a single, abundant enolase transcript on Northern (RNA) blots and a single protein on immunoblots, using antiserum raised against a recombinant enolase fusion protein. Because C. albicans enolase is abundantly produced during infection and elicits strong host immune responses, the mechanisms regulating enolase production are important for understanding the growth of C. albicans in vivo. To obtain more information on enolase gene expression by C. albicans, we used the enolase cDNA clone to investigate the genetic organization of enolase genes and the steady-state levels of enolase mRNA under several growth conditions. Gene disruption techniques in combination with Southern blot analyses of genomic DNA showed the presence of two enolase gene loci that could be distinguished by the locations of ClaI and Mn/I sites in their 3' flanking regions. Enolase steady-state mRNA levels were greatest during the middle phase of the logarithmic growth curve and were low during stationary phase. Minimal differences in enolase mRNA levels between yeast cells and hyphae were found. Propagation of C. albicans in glucose did not cause increased enolase mRNA levels compared with growth in a nonfermentable carbon source (pyruvate). It was concluded that two gene loci exist for C. albicans enolase and that enolase mRNA is constitutively produced at high levels during active metabolism. PMID:7896700

  13. Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing

    PubMed Central

    Torres, Tatiana Teixeira; Dolezal, Marlies; Schlötterer, Christian; Ottenwälder, Birgit

    2009-01-01

    Mitochondria play an essential role in several cellular processes. Nevertheless, very little is known about patterns of gene expression of genes encoded by the mitochondrial DNA (mtDNA). In this study, we used next-generation sequencing (NGS) for transcription profiling of genes encoded in the mitochondrial genome of Drosophila melanogaster and D. pseudoobscura. The analysis of males and females in both species indicated that the expression pattern was conserved between the two species, but differed significantly between both sexes. Interestingly, mRNA levels were not only different among genes encoded by separate transcription units, but also showed significant differences among genes located in the same transcription unit. Hence, mRNA abundance of genes encoded by mtDNA seems to be heavily modulated by post-transcriptional regulation. Finally, we also identified several transcripts with a noncanonical structure, suggesting that processing of mitochondrial transcripts may be more complex than previously assumed. PMID:19843606

  14. Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications.

    PubMed

    Moreno, Lilliana I; Tate, Courtney M; Knott, Erika L; McDaniel, Jade E; Rogers, Stephanie S; Koons, Barbara W; Kavlick, Mark F; Craig, Rhonda L; Robertson, James M

    2012-07-01

    The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood. PMID:22309221

  15. Kinetic models of the interference of gene transcription to ncRNA and mRNA

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-06-01

    The experiments indicate that the transcription of genes into ncRNA can positively or negatively interfere with transcription into mRNA. We propose two kinetic models describing this effect. The first model is focused on the ncRNA-induced chromatin modification facilitating the transcription of the downstream gene into mRNA. The second model includes the competition between the transcription into ncRNA and the binding of activator to a regulatory site of the downstream gene transcribed into mRNA. Our analysis based on the mean-field kinetic equations and Monte Carlo simulations shows the likely dependences of the transcription rate on RNA polymerase concentration in situations with different rate-limiting steps. Our models can also be used to scrutinize the dependence of the transcription rate on other kinetic parameters. Our kinetic Monte Carlo simulations show that the first model predicts stochastic bursts in the mRNA formation provided that the transcription into ncRNA is slow, while the second model predicts in addition anti-phase stochastic bursts in the mRNA and ncRNA formation provided that that the protein attachment to and detachment from a regulatory site is slow.

  16. mRNA as gene therapeutic: how to control protein expression.

    PubMed

    Tavernier, Geertrui; Andries, Oliwia; Demeester, Jo; Sanders, Niek N; De Smedt, Stefaan C; Rejman, Joanna

    2011-03-30

    For many years, it was generally accepted that mRNA is too unstable to be efficiently used for gene therapy purposes. In the last decade, however, several research groups faced this challenge and not only proved the feasibility of mRNA-mediated transfection with surprising results regarding transfection efficiency and duration of protein expression, but also were able to demonstrate major advantages over the use of pDNA. These advantages will be the first issue discussed in this review, which first of all addresses the notions that mRNA does not need to cross the nuclear barrier to exert its biological activity and in addition lacks CpG motifs, which reduces its immunogenicity. Secondly, it provides insight in the (in)stability of the mRNA molecule, in how mRNA can be modified to increase its half-life and in the necessities of exogenously produced mRNA to be successfully used in transfection protocols. Furthermore, this review gives an in-depth overview of the different techniques and vehicles for intracellular mRNA delivery exploited by us and other groups, comprising electroporation, gene gun injection, lipo- and polyplexes. Finally, it covers recent literature describing specific applications for mRNA based gene delivery, showing that until now most attention has been paid to vaccination strategies. This review offers a comprehensive overview of current knowledge of the major theoretical as well as practical aspects of mRNA-mediated transfection, showing both its possibilities and its pitfalls and should therefore be useful for a diverse scientific audience. PMID:20970469

  17. Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

    PubMed

    Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

    2011-09-01

    Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth. PMID:21725008

  18. Unification of gene expression data applying standard mRNA quantification references for comparable analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High throughput quantitative measurements of gene expression data have problems of reproducibility and comparability due to a lack of standard mRNA quantification references. Efforts have been made to safeguard data fidelity, yet generating quality expression data of inherent value remains a challe...

  19. Nonsense-mediated mRNA decay among coagulation factor genes

    PubMed Central

    Shahbazi, Shirin

    2016-01-01

    Objective(s): Haemostasis prevents blood loss following vascular injury. It depends on the unique concert of events involving platelets and specific blood proteins, known as coagulation factors. The clotting system requires precise regulation and coordinated reactions to maintain the integrity of the vasculature. Clotting insufficiency mostly occurs due to genetically inherited coagulation factor deficiencies such as hemophilia. Materials and Methods: A relevant literature search of PubMed was performed using the keywords coagulation factors, Nonsense-mediated mRNA decay and premature translation termination codons. Search limitations included English language and human-based studies. Results: Mutations that cause premature translation termination codons probably account for one-third of genetically inherited diseases. Transcripts bearing aberrant termination codons are selectively identified and eliminated by an evolutionarily conserved posttranscriptional pathway known as nonsense-mediated mRNA decay (NMD). There are many pieces of evidence of decay among coagulation factor genes. However, the hemophilia gene (F8) does not seem to be subjected to NMD. Since the F8 gene is located on the X-chromosome, a connection between X-linked traits and mRNA decay could be assumed. Conclusion: Considering that not all genes go through decay, this review focuses on the basics of the mechanism in coagulation genes. It is interesting to determine whether this translation-coupled surveillance system represents a general rule for the genes encoding components of the same physiological cascade. PMID:27279976

  20. Relationship of serotonin transporter gene polymorphisms and haplotypes to mRNA transcription.

    PubMed

    Bradley, Sarah L; Dodelzon, Katerina; Sandhu, Harinder K; Philibert, Robert A

    2005-07-01

    The serotonin transporter (5HTT; chromosomal location 17q12) is an important regulator of serotonergic neurotransmission and is the site of action for a number of antidepressant medications. Sequence variation at a VNTR known as the 5HTTLPR, which is 1.4 kb upstream of the translation start of 5HTT, has been associated in some studies with increased vulnerability to depression, neuroticism, and autism. Support for these clinical observations has included laboratory findings that 5HTTLPR variation is associated with changes in 5HTT gene translation. We re-examined these earlier laboratory findings by directly measuring 5HTT mRNA levels and genotyping four loci spanning the 5HTT gene using RNA and DNA prepared from 85 independent lymphoblast cell lines. Using this data, haplotypes were inferred and the resulting single point and haplotypes data analyzed by univariate and regression analyses. Consistent with the original findings, we found a significant effect of the 5HTTLPR on mRNA production. In contrast to previous reports, the effect on 5HTT mRNA production appeared to be mediated through an additive, not dominant, mechanism. Neither genotype nor haplotype at three other 5HTT loci were associated with alterations in mRNA production, although the small number of samples homozygous for the three most common haplotypes limits these findings. We conclude that further examination of the role of 5HTT sequence variation in regulating 5HTT mRNA production is warranted. PMID:15858822

  1. Embedding mRNA Stability in Correlation Analysis of Time-Series Gene Expression Data

    PubMed Central

    Farina, Lorenzo; De Santis, Alberto; Salvucci, Samanta; Morelli, Giorgio; Ruberti, Ida

    2008-01-01

    Current methods for the identification of putatively co-regulated genes directly from gene expression time profiles are based on the similarity of the time profile. Such association metrics, despite their central role in gene network inference and machine learning, have largely ignored the impact of dynamics or variation in mRNA stability. Here we introduce a simple, but powerful, new similarity metric called lead-lag R2 that successfully accounts for the properties of gene dynamics, including varying mRNA degradation and delays. Using yeast cell-cycle time-series gene expression data, we demonstrate that the predictive power of lead-lag R2 for the identification of co-regulated genes is significantly higher than that of standard similarity measures, thus allowing the selection of a large number of entirely new putatively co-regulated genes. Furthermore, the lead-lag metric can also be used to uncover the relationship between gene expression time-series and the dynamics of formation of multiple protein complexes. Remarkably, we found a high lead-lag R2 value among genes coding for a transient complex. PMID:18670596

  2. hnRNP-U enhances the expression of specific genes by stabilizing mRNA.

    PubMed

    Yugami, Masato; Kabe, Yasuaki; Yamaguchi, Yuki; Wada, Tadashi; Handa, Hiroshi

    2007-01-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to be involved in pre-mRNA processing. hnRNP-U, also termed scaffold attachment factor A (SAF-A), binds to pre-mRNA and nuclear matrix/scaffold attachment region DNA elements. However, its role in the regulation of gene expression is as yet poorly understood. In the present study, we show that hnRNP-U specifically enhances the expression of tumor necrosis factor alpha mRNA by increasing its stability, possibly through binding to the 3' untranslated region. We also show that hnRNP-U enhances the expression of several other genes as well, including GADD45A, HEXIM1, HOXA2, IER3, NHLH2, and ZFY, by binding to and stabilizing these mRNAs. These results suggest that hnRNP-U enhances the expression of specific genes by regulating mRNA stability. PMID:17174306

  3. Comparison of Protamine 1 to Protamine 2 mRNA Ratio and YBX2 gene mRNA Content in Testicular Tissue of Fertile and Azoospermic Men

    PubMed Central

    Moghbelinejad, Sahar; Najafipour, Reza; Hashjin, Amir Samimi

    2015-01-01

    Background Although aberrant protamine (PRM) ratios have been observed in infertile men, the mechanisms that implicit the uncoupling of PRM1 and PRM2 expression remain unclear. To uncover these mechanisms, in this observational study we have compared the PRM1/PRM2 mRNA ratio and mRNA contents of two regulatory factors of these genes. Materials and Methods In this experimental study, sampling was performed by a multi-step method from 50 non-obstructive azoospermic and 12 normal men. After RNA extraction and cDNA synthesis, real-time quantitative polymerase chain reaction (RT- QPCR) was used to analyze the PRM1, PRM2, Y box binding protein 2 (YBX2) and JmjC-containing histone demethylase 2a (JHDM2A) genes in testicular biopsies of the studied samples. Results The PRM1/PRM2 mRNA ratio differed significantly among studied groups, namely 0.21 ± 0.13 in azoospermic samples and -0.8 ± 0.22 in fertile samples. The amount of PRM2 mRNA, significantly reduced in azoospermic patients. Azoospermic men exhibited significant under expression of YBX2 gene compared to controls (P<0.001). mRNA content of this gene showed a positive correlation with PRM mRNA ratio (R=0.6, P=0.007). JHDM2A gene expression ratio did not show any significant difference between the studied groups (P=0.3). We also observed no correlation between JHDM2A mRNA content and the PRM mRNA ratio (R=0.2, P=0.3). Conclusion We found significant correlation between the aberrant PRM ratio (PRM2 under expression) and lower YBX2 mRNA content in testicular biopsies of azoospermic men compared to controls, which suggested that downregulation of the YBX2 gene might be involved in PRM2 under expression. These molecules could be useful biomarkers for predicting male infertility. PMID:26644857

  4. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    NASA Astrophysics Data System (ADS)

    Elgart, Vlad; Jia, Tao; Fenley, Andrew T.; Kulkarni, Rahul

    2011-08-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distribution. Additionally, if gene expression is repressed such that observed protein bursts arise only from single mRNAs, we show how observations of protein burst distributions (repressed and unrepressed) can be used to completely determine the mRNA burst distribution. Assuming independent contributions from individual bursts, we derive analytical expressions connecting means and variances for burst and steady-state protein distributions. Finally, we validate our general analytical results by considering a specific reaction scheme involving regulation of protein bursts by small RNAs. For a range of parameters, we derive analytical expressions for regulated protein distributions that are validated using stochastic simulations. The analytical results obtained in this work can thus serve as useful inputs for a broad range of studies focusing on stochasticity in gene expression.

  5. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

    PubMed Central

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

  6. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  7. mRNA trans-splicing in gene therapy for genetic diseases.

    PubMed

    Berger, Adeline; Maire, Séverine; Gaillard, Marie-Claude; Sahel, José-Alain; Hantraye, Philippe; Bemelmans, Alexis-Pierre

    2016-07-01

    Spliceosome-mediated RNA trans-splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post-transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre-mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans-splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans-splicing, review the different strategies that are under evaluation to lead to efficient trans-splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487-498. doi: 10.1002/wrna.1347 For further resources related to this article, please visit the WIREs website. PMID:27018401

  8. Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

    PubMed Central

    Marguerat, Samuel; Lawler, Katherine; Brazma, Alvis; Bähler, Jürg

    2014-01-01

    The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress. PMID:25007214

  9. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

    PubMed

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-05-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

  10. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues

    PubMed Central

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-01-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

  11. Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene

    SciTech Connect

    Cybulsky, M.I.; Fries, J.W.U.; Williams, A.J.; Sultan, P.; Gimbrone, M.A. Jr.; Collins, T. ); Eddy, R.; Byers, M.; Shows, T. )

    1991-09-01

    Vascular cell adhesion molecule 1 (VCAM-1) is a cell surface glycoprotein adhesive for certain blood leukocytes and tumor cells, which is expressed by activated endothelium in a variety of pathologic conditions including atherosclerosis. Genomic clones encoding the VCAM1 gene were isolated and the organization of the gene was determined. The gene, which is present in a single copy in the human genome, contains 9 exons spanning {approx}25 kilobases of DNA. Exons 2-8 contain C2 or H-type immunoglobulin domains. At least two different VCAM-1 precursors can be generated from the human gene as a result of alternative mRNA splicing events, which include or exclude exon 5. A consensus TATAA element is located upstream of the transcriptional start site. The VCAM1 promoter contains consensus binding sites for NF-{kappa}B, the GATA family of transcription factors, as well as an AP1 site. The VCAM1 gene was assigned to the 1p31-32 region of chromosome 1 based on the analysis of human-mouse hybrid cell lines and in situ hybridization. Structural analysis of the human VCAM1 gene provides the basis for alternative mRNA splicing and an initial approach to elucidating the regulation of VCAM-1 expression.

  12. mRNA expression profiles of calmodulin and liver receptor homolog-1 genes in chickens.

    PubMed

    Zhang, Z-C; Xiao, L-H; Wang, Y; Chen, S-Y; Yang, Z-Q; Zhao, X-L; Zhu, Q; Liu, Y-P

    2012-01-01

    Calmodulin (CALM), a calcium-binding protein, is expressed in the hypothalamic-pituitary-gonadal axis; it plays a pivotal role in the reproductive system by regulating gonadotropin-releasing hormone signaling. Downstream of hypothalamic-pituitary-gonadal signaling pathways, liver receptor homolog-1 (LRH-1) is involved in female gonadal hormone synthesis. In the chicken, although the two genes are known to be associated with reproductive traits, the interaction between gonadotropins and gonadal steroids remains unclear. We used quantitative real-time PCR to quantify the tissular (hypothalamus, pituitary, ovary, liver, kidney, oviduct, heart) and ontogenetic (12, 18, 32, and 45 weeks) mRNA expression profiles of CALM and LRH-1 in Erlang Mountainous chickens to determine their roles in the endocrine control of fertility, and compared these profiles with expression in Roman chickens. We found that the relative expressions of CALM and LRH-1 genes had the highest levels in the pituitary and ovary at 32 weeks. The expression level of CALM mRNA in the pituitary of Roman chickens was significantly higher than that in Erlang Mountainous chickens at 32 and 45 weeks, while the LRH-1 transcript level in the ovaries of Roman chickens was significantly lower than that of Erlang Mountainous chickens at 32 and 45 weeks. In summary, the transcript levels of CALM and LRH-1 genes are associated with chicken reproductive traits; in addition, we found that the CALM gene is the key regulator in the hypothalamic-pituitary-gonadal signaling network. PMID:23079841

  13. Genomic Analysis and mRNA Expression of Equine Type I Interferon Genes

    PubMed Central

    Detournay, Olivier; Morrison, David A.; Wagner, Bettina; Zarnegar, Behdad

    2013-01-01

    This study aimed at identifying all of the type I interferon (IFN) genes of the horse and at monitoring their expression in equine cells on in vitro induction. We identified 32 putative type I IFN loci on horse chromosome 23 and an unplaced genomic scaffold. A phylogentic analysis characterized these into 8 different type I IFN classes, that is, putative functional genes for 6 IFN-α, 4 IFN-β, 8 IFN-ω (plus 4 pseudogenes), 3 IFN-δ (plus 1 pseudogene), 1 IFN-κ and 1 IFN-ɛ, plus 1 IFN-ν pseudogene, and 3 loci belonging to what has previously been called IFN-αω. Our analyses indicate that the IFN-αω genes are quite distinct from both IFN-α and IFN-ω, and we refer to this type I IFN as IFN-μ. Results from cell cultures showed that leukocytes readily expressed IFN-α, IFN-β, IFN-δ, IFN-μ, and IFN-ω mRNA on induction with, for example, live virus; while fibroblasts only expressed IFN-β mRNA on stimulation. IFN-κ or IFN-ɛ expression was not consistently induced in these cell cultures. Thus, the equine type I IFN family comprised 8 classes, 7 of which had putative functional genes, and mRNA expression of 5 was induced in vitro. Moreover, a relatively low number of IFN-α subtypes was found in the horse compared with other eutherian mammals. PMID:23772953

  14. Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis.

    PubMed

    Brosnan, C A; Mitter, N; Christie, M; Smith, N A; Waterhouse, P M; Carroll, B J

    2007-09-11

    In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1-8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission. PMID:17785412

  15. RNA editing by G-nucleotide insertion in mumps virus P-gene mRNA transcripts.

    PubMed Central

    Paterson, R G; Lamb, R A

    1990-01-01

    A guanine nucleotide insertion event has been shown to occur at a specific site within mumps virus P-gene mRNA transcripts. The region of the mRNA containing the site expected to be used for RNA editing and the complementary portion of the genomic RNA were cloned, and their nucleotide sequences were obtained. The genomic RNA was found to possess six C residues at the insertion site, whereas 63% of the P-gene-specific mRNA transcripts were found to have from two to five G residues inserted at this position in the RNA. An unedited mRNA was shown to encode the mumps virus cysteine-rich protein V, and mRNA transcripts containing two and four inserted G residues were translated to yield the mumps virus P and I proteins, respectively. Images PMID:2166809

  16. DNA Micelle Flares for Intracellular mRNA Imaging and Gene Therapy

    PubMed Central

    Chen, Tao; Sam Wu, Cuichen; Jimenez, Elizabeth; Zhu, Zhi; Dajac, Joshua G.; You, Mingxu; Han, Da

    2013-01-01

    Multifunctional DNA micelles: Molecular beacon micelle flares (MBMFs), based on diacyllipid-molecular beacon conjugate (L-MB) self-assembly, have been developed for combined mRNA detection and gene therapy. The advantages of these micelle flares include easy probe synthesis, efficient cellular uptake, enhanced enzymatic stability, high signal-to-background ratio, excellent target selectivity, and superior biocompatibility. In addition, these probes possess a hydrophobic cavity that can be used for additional hydrophobic agents, holding great promise for constructing an all-in-one nucleic acid probe. PMID:23319350

  17. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    PubMed Central

    Eskandari-Nasab, Ebrahim; Hashemi, Mohammad; Rafighdoost, Firoozeh

    2016-01-01

    Background. Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent nonneoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent nonneoplastic tissue (OR = 2.30, 95% CI = 0.95–5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P = 0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients' clinical characteristics (P > 0.05). Conclusion. Our findings confirmed upregulation of RGC32 in breast cancerous tumors, but it was not associated with promoter methylation patterns. PMID:27118972

  18. Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display

    PubMed Central

    Li, Xiang; Bi, Zhenghong; Di, Rong; Liang, Peng; He, Qiguang; Liu, Wenbo; Miao, Weiguo; Zheng, Fucong

    2016-01-01

    Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant–pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew. PMID:26840302

  19. Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display.

    PubMed

    Li, Xiang; Bi, Zhenghong; Di, Rong; Liang, Peng; He, Qiguang; Liu, Wenbo; Miao, Weiguo; Zheng, Fucong

    2016-01-01

    Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant-pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew. PMID:26840302

  20. Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display.

    PubMed

    Lang, Ping; Zhang, Can-Kui; Ebel, Robert C; Dane, Fenny; Dozier, William A

    2005-10-10

    Citrus unshiu is freeze tolerant to -10 degrees C when fully acclimated after exposure to cold, nonfreezing temperatures. To gain an understanding of its cold tolerance mechanism, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) and quantitative relative RT-PCR were used to study gene expression under a gradual cold-acclimation temperature regime. Six up-regulated and two down regulated genes were identified based on their amino acid sequences. The identified proteins encoded by the up-regulated genes were: 14-3-3 protein, 40S ribosomal protein S23, putative 60S ribosomal protein L15, nucleoside diphosphate kinase III protein, regulator of chromosome condensation-like protein, and amino acid permease 6. The proteins encoded by the two down-regulated genes were: miraculin-like protein and beta-galactosidase. Their individual function has been briefly reviewed based on published information. In addition to the findings in this study, we compared the function of cold responsive genes of Poncirus trifoliata, a very cold hardy relative of Citrus species that is freeze tolerant to -30 degrees C when fully acclimated, to the function of genes in the current study. PMID:16125877

  1. Herpes simplex virus infection selectively stimulates accumulation of beta interferon reporter gene mRNA by a posttranscriptional mechanism.

    PubMed Central

    Mosca, J D; Pitha, P M; Hayward, G S

    1992-01-01

    To study the mechanism of a novel herpes simplex virus (HSV) activity that stimulates expression of reporter genes containing beta interferon (IFN-beta)-coding sequences, we have established permanent DNA-transfected cell lines that each contain two distinct hybrid genes encoding mRNA species with different half-lives. These reporter genes comprised either the human IFN-beta- or bacterial chloramphenicol acetyltransferase (CAT)-coding and 3' untranslated regions placed under the transcriptional control of the powerful major immediate-early promoter-enhancer region (IE94) from simian cytomegalovirus. Most of the dual-transfected cell lines yielded significant levels of steady-state IE94-CAT mRNA and abundant constitutive synthesis of CAT enzyme activity, whereas no accumulation of IE94-IFN mRNA could be detected. However, infection with HSV type 1 resulted in a 300-fold increase in IE94-IFN-specific mRNA transcripts, compared with no more than 3- to 5-fold stimulation of IE94-CAT-specific mRNA. In contrast, cycloheximide treatment increased stable mRNA levels and transcription initiation rates from both the IE94-IFN and IE94-CAT hybrid genes. Run-on transcription assays in isolated nuclei suggested that induction of IE94-IFN gene expression by HSV type 1 occurred predominantly at the posttranscriptional level. Enhancement of the unstable IFN mRNA species after HSV infection was also observed in cell lines containing a simian virus 40 enhancer-driven IFN gene (SV2-IFN). Similarly, in transient-transfection assays, both SV2-IFN and IE94-IFN gave only low basal mRNA synthesis, but superinfection with HSV again led to high-level accumulation of IFN mRNA. Finally, substitution of the SV2-IFN gene 3' region with poly(A) and splicing signals from the SV2-CAT gene cassette led to stabilization of the IFN mRNA even in the absence of HSV. Therefore, we conclude that HSV infection leads to selective accumulation of IFN-beta mRNA by a posttranscriptional mechanism that is

  2. Immunochemical isolation of gamma-globulin mRNA and estimation of immunoglobulin gene reiteration.

    PubMed

    Muto, M

    1977-01-01

    The polyribosomes synthesizing gamma-globulin have been isolated by the achievement of specific precipitation using bentonite-treated anti-IgG antibody. The RNA extracted from the immunochemically precipitated polysomes was tested for its ability to direct the synthesis of proteins in a cell-free system. The specific gamma-globulin-synthesizing activity (cpm of gamma-globulin synthesized/microgram RNA) of this RNA was 10-fold greater than that from total polysomes. gamma-globulin mRNA (messenger RNA) isolated by immunoprecipitation was more than 89% pure with respect to contamination by other species of mRNA. The products synthesized by the cell-free system were also analyzed by sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis. This RNA has been hybridized with mouse myeloma DNA. The estimation of immunoglobulin gene reiteration was carried out using hybridization kinetics with consideration given to the DNA/RNA ratio since the estimation from the "half Cot value" is not accurate. The results suggest that in the mouse there are about 20 copies per subgroup of genes coding for the variable region of the H and L chains. PMID:927224

  3. Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation.

    PubMed Central

    el-Mahdani, N.; Vaillant, J. C.; Guiguet, M.; Prévot, S.; Bertrand, V.; Bernard, C.; Parc, R.; Béréziat, G.; Hermelin, B.

    1997-01-01

    We analysed the frequency of p53 mRNA overexpression in a series of 109 primary colorectal carcinomas and its association with p53 gene mutation, which has been correlated with short survival. Sixty-nine of the 109 cases (63%) demonstrated p53 mRNA overexpression, without any correlation with stage or site of disease. Comparison with p53 gene mutation indicated that, besides cases in which p53 gene mutation and p53 mRNA overexpression were either both present (40 cases) or both absent (36 cases), there were also cases in which p53 mRNA was overexpressed in the absence of any mutation (29 cases) and those with a mutant gene in which the mRNA was not overexpressed (four cases). Moreover, the mutant p53 tumours exhibited an increase of p53 mRNA expression, which was significantly higher in tumours expressing the mutated allele alone than in tumours expressing both wild- and mutated-type alleles. These data (1) show that p53 mRNA overexpression is a frequent event in colorectal tumours and is not predictive of the status of the gene, i.e. whether or not a mutation is present; (2) provide further evidence that p53 protein overexpression does not only result from an increase in the half-life of mutated p53 and suggest that inactivation of the p53 function in colorectal cancers involves at least two distinct mechanisms, including p53 overexpression and/or mutation; and (3) suggest that p53 mRNA overexpression is an early event, since it is not correlated with Dukes stage. PMID:9052405

  4. mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

    PubMed

    Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

    2015-06-01

    The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme. PMID:25963717

  5. The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons

    PubMed Central

    García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A.; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E.

    2016-01-01

    We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay. The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. PMID:26717982

  6. The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

    PubMed

    García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

    2016-05-01

    We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. PMID:26717982

  7. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  8. No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

    PubMed Central

    Farace, M G; Aellen, M F; Briand, P A; Faust, C H; Vassalli, P; Mach, B

    1976-01-01

    RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed. Images PMID:815907

  9. Molecular cloning and mRNA expression analysis of sheep MYL3 and MYL4 genes.

    PubMed

    Zhang, Chunlan; Wang, Jianmin; Wang, Guizhi; Ji, Zhibin; Hou, Lei; Liu, Zhaohua; Chao, Tianle

    2016-02-15

    Using longissimus dorsi muscles of Dorper sheep as the experimental materials, the complete cDNAs of ovine MYL3 (Myosin light chain 3) and MYL4 (Myosin light chain 4) genes were cloned using RT-PCR, 5' RACE and 3' RACE. We obtained 925-bp and 869-bp full-length cDNAs and submitted their sequences to GenBank as accession numbers of KJ710703 and KJ768855, respectively. The cDNAs contained 600-bp and 582-bp open reading frames (ORFs) and encoded proteins comprising 199 and 193 amino acid residues, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation, O-glycosylation, and phosphorylation sites. The secondary structures of MYL3 and MYL4 were predicted to be 40.70% and 48.70% α- helical, respectively. Sequence alignment showed that the MYL3 and MYL4 proteins of Ovis aries both shared more than 91% amino acid sequence similarity with those of Mus musculus, Homo sapiens, Rattus norvegicus, Bos taurus, and Sus scrofa. The levels of MYL3 and MYL4 mRNA in various sheep tissues were determined using qRT-PCR. The results showed that both mRNAs were highly expressed in the heart. This study has established a foundation for further investigation of the ovine MYL3 and MYL4 genes. PMID:26656596

  10. Isolation of genes involved in colorectal metastasis by differential display of mRNA species

    SciTech Connect

    Gustafson, C.; Chenevix-Trench, G.; Antalis, T.

    1994-09-01

    The genetic events that give rise to malignant colorectal tumors have been determined in some detail. Much less is known about the genes involved in metastasis of these neoplasms. A useful resource to study this process is the pair of cell lines, SW480 and SW620, which are derived from the primary and metastatic components, respectively, of the same colorectal tumor. We are using the method of differential display of mRNA species to isolate genes that are differentially expressed in these two cell lines. Differential display is carried out in triplicate, using three different RNA extractions from each cell line. Only fragments that are consistently up- or down-regulated in one cell line compared to another are examined further. Less than 1% of fragments are differentially expressed. These are cloned, sequenced, and used for Northern blot and reverse transcriptase-PCR in order to examine their differential expression further. The RNA sources for this expression analysis are (i) SW480 and SW620 cells, (ii) other pairs of primary and metastatic colorectal cell lines, (iii) primary and metastatic tissue from patients with colorectal cancer.

  11. The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

    PubMed

    Scholthof, H B; Gowda, S; Wu, F C; Shepherd, R J

    1992-05-01

    Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes. PMID:1560539

  12. Reduced XPC DNA repair gene mRNA levels in clinically normal parents of xeroderma pigmentosum patients.

    PubMed

    Khan, Sikandar G; Oh, Kyu-Seon; Shahlavi, Tala; Ueda, Takahiro; Busch, David B; Inui, Hiroki; Emmert, Steffen; Imoto, Kyoko; Muniz-Medina, Vanessa; Baker, Carl C; DiGiovanna, John J; Schmidt, Deborah; Khadavi, Arash; Metin, Ahmet; Gozukara, Engin; Slor, Hanoch; Sarasin, Alain; Kraemer, Kenneth H

    2006-01-01

    Xeroderma pigmentosum group C (XP-C) is a rare autosomal recessive disorder. Patients with two mutant alleles of the XPC DNA repair gene have sun sensitivity and a 1000-fold increase in skin cancers. Clinically normal parents of XP-C patients have one mutant allele and one normal allele. As a step toward evaluating cancer risk in these XPC heterozygotes we characterized cells from 16 XP families. We identified 15 causative mutations (5 frameshift, 6 nonsense and 4 splicing) in the XPC gene in cells from 16 XP probands. All had premature termination codons (PTC) and absence of normal XPC protein on western blotting. The cell lines from 26 parents were heterozygous for the same mutations. We employed a real-time quantitative reverse transcriptase-PCR assay as a rapid and sensitive method to measure XPC mRNA levels. The mean XPC mRNA levels in the cell lines from the XP-C probands were 24% (P<10(-7)) of that in 10 normal controls. This reduced XPC mRNA level in cells from XP-C patients was caused by the PTC that induces nonsense-mediated mRNA decay. The mean XPC mRNA levels in cell lines from the heterozygous XP-C carriers were intermediate (59%, P=10(-4)) between the values for the XP patients and the normal controls. This study demonstrates reduced XPC mRNA levels in XP-C patients and heterozygotes. Thus, XPC mRNA levels may be evaluated as a marker of cancer susceptibility in carriers of mutations in the XPC gene. PMID:16081512

  13. mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

    PubMed

    Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

    2010-05-01

    Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system. PMID:20360389

  14. Brain-derived peptides increase blood-brain barrier GLUT1 glucose transporter gene expression via mRNA stabilization.

    PubMed

    Boado, R J

    1998-10-23

    The present investigation studied the effect of the brain-derived peptide preparation Cerebrolysin (CI, EBEWE, Austria) on the turnover rate and gene expression of the blood-brain barrier (BBB) GLUT1 glucose transporter mRNA. Studies were performed in brain endothelial cultured cells transfected with the human (h) GLUT1 transcript. In control cells, the full length 2.8 Kb hGLUT1 mRNA was rapidly degraded following transfection, and the abundance of this transcript at 4 and 6 h was comparable to background mRNA levels seen in cells transfected without hGLUT1 mRNA. On the contrary, the decay of the hGLUT1 mRNA was stabilized in CI-treated cells resulting in a marked reduction in the fractional turnover rate (72.4 and 4.0%/h, control and CI, respectively). In parallel experiments, CI induced a significant increase in the levels of immunoreactive GLUT1 protein measured by enzyme-linked immunosorbent assay (ELISA). In conclusion, data presented here demonstrate that factors in CI increase BBB-GLUT1 transcript stability, and that this is associated with an induction of BBB-GLUT1 gene expression in brain endothelial cultured cells. PMID:9832194

  15. Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies.

    PubMed

    Greger, D L; Gropp, F; Morel, C; Sauter, S; Blum, J W

    2006-11-01

    Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARalpha, PPARgamma) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n=7 per group). Upon first presentation of dogs, mRNA levels of PPARalpha, PPARgamma, CAR, PXR and RXRalpha in duodenum as well as PPARgamma, CAR, PXR and RXRalpha in colon were not different among groups (P>0.10). Although mRNA abundance of PPARalpha in colon of dogs with FRD was similar in both IBD and CON (P>0.10), PPARalpha mRNA abundance was higher in IBD than CON (P<0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P<0.05) or CON (P<0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P<0.05). Differences in mRNA levels of PPARalpha and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD. PMID:16446074

  16. In Vitro Synthesis, Delivery, and Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac Transposition.

    PubMed

    Bire, Solenne; Ishac, Nicole; Rouleux-Bonnin, Florence

    2016-01-01

    Nowadays, nonviral gene transfer is currently of great importance for introducing exogenous genes into genomes and for ensuring that transgene expression is suitable for therapeutic and bioproduction purposes. The piggyBac transposon-based system is particularly interesting since it is easy to engineer and has a large cargo capacity, up to 100 kb. In its setup, the system requires only the piggyBac transposase protein and the transgene delineated by the two piggyBac-specific inverted terminal repeats. Usually the source of transposase is carried by a DNA plasmid. However, the principal drawback of this method is the lasting presence of the transposase, due to episomal persistence or possible integration of the transposase gene vector into the cell's genome. This can lead to genotoxic effects such as multiple genomic integration events and remobilization of the transposon vector once it has been integrated. One alternative to improve the safety of the system is to deliver the transposase as in vitro-synthesized messenger RNA in order to define a very narrow expression window during which a one-shot transposition process would occur. Issues that can be encountered when working on mRNA cell transfer are related to the quality of the synthetic mRNA, the system used to introduce mRNA into the cells and the bioavailability of the mRNA molecules. Here we describe a method to produce mRNA, verify its quality, determine which transfecting reagents can be used and how this mRNA is available to promote the transposition process in HeLa cells. Additionally, we illustrate this method in stromal mesenchymal cell lines in order to support hematopoiesis. PMID:27236801

  17. Characterization of the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast

    SciTech Connect

    Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We cloned the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast. Black-Right-Pointing-Pointer The ptr5{sup +} gene was found to encode nucleoporin 85 (Nup85). Black-Right-Pointing-Pointer Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. Black-Right-Pointing-Pointer Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. Black-Right-Pointing-Pointer Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A){sup +} RNA transport] 1 to 11, which accumulate poly(A){sup +} RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5-1 mutant shows dots- or a ring-like accumulation of poly(A){sup +} RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5{sup +} gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5-1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5-1 mutation. In addition, we found that the ptr5-1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5-1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

  18. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  19. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing

    SciTech Connect

    Kaye, F.; Battey, J.; Nau, M.; Brooks, B.; Seifter, E.; De Greve, J.; Birrer, M.; Sausville, E.; Minna, J.

    1988-01-01

    The authors' analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs that is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

  20. mRNA Noise Reveals that Activators Induce a Biphasic Response in the Promoter Kinetics of Highly Regulated Genes

    NASA Astrophysics Data System (ADS)

    Quinn, Katie; To, Tsz-Leung; Maheshri, Narendra

    2012-02-01

    A dominant source of fluctuations in gene expression is thought to be the process of transcription. The statistics of these fluctuations arise from the kinetics of transcription. Multiple studies suggest the bulk of fluctuations can be understood by a simple process where genes are inactive for exponentially distributed times punctuated by geometric bursts of mRNA. Yet it's largely unknown how cis and trans factors affect the two lumped kinetic parameters, burst size and burst frequency, that describe this process. Importantly, how these parameters are regulated in a single gene can qualitatively affect the dynamical behavior of the network it is embedded within. Here, we ask whether transcriptional activators increase gene expression by increasing the burst size or burst frequency. We do so by deducing these parameters from steady-state mRNA distributions measured in individual yeast cells using single molecule mRNA FISH. We find that for both a synthetic and natural promoter, activators appear to first increase burst size, then burst frequency. We suggest this biphasic response may be common to all highly regulated genes and was previously unappreciated because of measurement techniques. Furthermore, its origins appear to relate to cis events at the promoter, and may arise from combinations of basal and activator-dependent bursts. Our measurements shed new light on transcriptional mechanisms and should assist in building synthetic promoters with tunable statistics.

  1. The use of logic relationships to model colon cancer gene expression networks with mRNA microarray data.

    PubMed

    Ruan, Xiaogang; Wang, Jinlian; Li, Hui; Perozzi, Rhoda E; Perozzi, Edmund F

    2008-08-01

    The ultimate goal of genomics research is to describe the network of molecules and interactions that govern all biological functions and disease processes in cells. Nonlinear interactions among genes in terms of their logic relationships play a key role for deciphering the networks of molecules that underlie cellular function. We present a method based on a graph coloring scheme and information theory to identify the gene expression network with lower and higher order logic interactions of genes. The analysis of oncogenes and suppressor genes from a colon cancer mRNA microarray dataset identifies a gene expression network with directionality and weights that reflects intracellular communication pathways. The success of the proposed method in mining hidden, complicated gene interactions and reliably interpreting experimental results suggests that the proposed method is a useful tool for understanding cancer systems. Extension of this method holds the potential to be fruitful for understanding other complex, nonsymmetric systems. PMID:18249040

  2. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  3. mRNA export protein THOC5 as a tool for identification of target genes for cancer therapy.

    PubMed

    Tran, Doan Duy Hai; Saran, Shashank; Koch, Alexandra; Tamura, Teruko

    2016-04-10

    Recent evidence indicates that mRNA export is selective, giving priority to a subset of mRNAs that control diverse biological processes including cell proliferation, differentiation, stress response, and cell survival as well as tumor development. The depletion of a member of the mRNA export complex, the THO complex, impairs the expression of only a subset of genes, but causes dramatic changes in phenotype, such as cell cycle inhibition, abnormal differentiation, and importantly apoptosis of stem cells and cancer cells but not normal epithelial cells, hepatocytes, or fibroblasts. Recent exosome sequence data revealed that over 100 driver gene mutations with a number of signaling pathways are involved in human cancer formation, indicating that multiple signaling pathways will need to be inhibited for cancer therapy. In this review we firstly describe a basic feature and function of the mRNA export complex, THO, secondly, the biological alteration upon depletion of a member of the THO complex in normal and cancer cells, and thirdly, identification of its target genes. Finally we describe our recent data on selection of targeting candidates from THOC5 dependent genes for application in cancer therapy. PMID:26828015

  4. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    PubMed Central

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  5. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    PubMed Central

    Thomsen, Rune; Daugaard, Tina F.; Holm, Ida E.; Nielsen, Anders Lade

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease. PMID:23991052

  6. BayMiR: inferring evidence for endogenous miRNA-induced gene repression from mRNA expression profiles

    PubMed Central

    2013-01-01

    Background Popular miRNA target prediction techniques use sequence features to determine the functional miRNA target sites. These techniques commonly ignore the cellular conditions in which miRNAs interact with their targets in vivo. Gene expression data are rich resources that can complement sequence features to take into account the context dependency of miRNAs. Results We introduce BayMiR, a new computational method, that predicts the functionality of potential miRNA target sites using the activity level of the miRNAs inferred from genome-wide mRNA expression profiles. We also found that mRNA expression variation can be used as another predictor of functional miRNA targets. We benchmarked BayMiR, the expression variation, Cometa, and the TargetScan “context scores” on two tasks: predicting independently validated miRNA targets and predicting the decrease in mRNA abundance in miRNA overexpression assays. BayMiR performed better than all other methods in both benchmarks and, surprisingly, the variation index performed better than Cometa and some individual determinants of the TargetScan context scores. Furthermore, BayMiR predicted miRNA target sets are more consistently annotated with GO and KEGG terms than similar sized random subsets of genes with conserved miRNA seed regions. BayMiR gives higher scores to target sites residing near the poly(A) tail which strongly favors mRNA degradation using poly(A) shortening. Our work also suggests that modeling multiplicative interactions among miRNAs is important to predict endogenous mRNA targets. Conclusions We develop a new computational method for predicting the target mRNAs of miRNAs. BayMiR applies a large number of mRNA expression profiles and successfully identifies the mRNA targets and miRNA activities without using miRNA expression data. The BayMiR package is publicly available and can be readily applied to any mRNA expression data sets. PMID:24001276

  7. Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes.

    PubMed

    Kar, Parmita; Chawla, Himika; Saha, Soma; Tandon, Nikhil; Goswami, Ravinder

    2016-06-01

    Selection of appropriate housekeeping-genes as reference is important in mRNA expression-related experiments. It is more important in diabetes since hyperglycemia per se can influence expression of housekeeping-genes. RNA expression of Glyceraldehyde-3-phosphate-dehydrogenase, β-actin and 18S-ribosomal-RNA, Hypoxanthine-phosphoribosyl-transferase (HPRT), Tyrosine-3-monooxygenase/tryptophan (YHWAZ), β2-microglobin (β2M), TATA-binding-protein (TBP), and Ubiquitin C and cytochrome1 (CYC1) assessed in circulating-lymphocytes-(PBMC) of patients with type-1-diabetes and healthy controls. The stability ('M' value <1.02) and number of housekeeping-genes required for normalization in qRT-PCR were determined by 'ge-norm software.' Vitamin-D-receptor (VDR) was used as a target gene. All the nine genes tested had sufficient 'M' value in diabetes and healthy controls. However, housekeeping-genes indicated a relatively higher stability of expression in healthy controls in comparison to diabetes. Use of single housekeeping-genes brought gross variation in the calculation of VDR-mRNA copies. The ge-norm software suggested geometric mean of five housekeeping-genes for ideal normalization in diabetes (CYC1, β-actin, YHWAZ, HPRT, and β2M) and only three in controls (CYC1, β-actin, and TBP). HbA1c did not correlate with expression of any of the nine housekeeping-genes. Thus, geometric mean of CYC1, β-actin, YHWAZ, HPRT, and β2M needs to be used for ideal normalization of mRNA in type-1-diabetes. Similar studies are required in other population. PMID:27160934

  8. Effect of propionate on mRNA expression of key genes for gluconeogenesis in liver of dairy cattle.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Bequette, Brian J; Donkin, Shawn S

    2015-12-01

    glucagon in calves receiving propionate were not different than controls. Calves receiving propionate had increased PCK1 mRNA, tended to have increased G6PC mRNA, and had similar PC mRNA compared with saline controls. These data indicate a tendency for in vivo effects of propionate to alter hepatic gene expression in mid-lactation cows and neonatal calves, which are consistent with a feed-forward effect of propionate to regulate its own metabolism toward gluconeogenesis through changes in hepatic PCK1 mRNA. PMID:26409969

  9. A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR

    PubMed Central

    Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

    2016-01-01

    Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

  10. Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

    USGS Publications Warehouse

    Yada, T.; Hyodo, S.; Schreck, C.B.

    2008-01-01

    Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems. ?? 2008 Elsevier Inc. All rights reserved.

  11. Characterization and mRNA expression profile of the TbNre1 gene of the ectomycorrhizal fungus Tuber borchii.

    PubMed

    Guescini, Michele; Stocchi, L; Sisti, D; Zeppa, S; Polidori, E; Ceccaroli, P; Saltarelli, R; Stocchi, V

    2009-02-01

    This study focuses on the cloning and characterization of the major nitrogen regulator element from the ectomycorrhizal fungus Tuber borchii, TbNre1. Sequence analysis of the predicted protein and complementation experiments in Neurospora crassa demonstrated that the cloned gene is orthologous to areA/nit-2 gene. Transcriptional expression investigations by real-time RT-PCR showed TbNre1 up-regulation in the presence of nitrate or in the absence of nitrogen during free-living mycelium growth. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred nitrogen sources like ammonium and glutamine. Furthermore, TbNre1 mRNA was found to be up-regulated during T. borchii and T. platyphyllos interaction. All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in the free living mycelium and in T. borchii-T. platyphyllos interaction. Finally, the possible role of the transcription factor TBNRE1 in the induction of proteases and virulence-like genes, necessary in ectomycorrhizal establishment, was also discussed. PMID:19115055

  12. Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

    SciTech Connect

    Grzybowska, Ewa A.

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Functional characteristics of intronless genes (IGs). Black-Right-Pointing-Pointer Diseases associated with IGs. Black-Right-Pointing-Pointer Origin and evolution of IGs. Black-Right-Pointing-Pointer mRNA processing without splicing. -- Abstract: Intronless genes (IGs) constitute approximately 3% of the human genome. Human IGs are essentially different in evolution and functionality from the IGs of unicellular eukaryotes, which represent the majority in their genomes. Functional analysis of IGs has revealed a massive over-representation of signal transduction genes and genes encoding regulatory proteins important for growth, proliferation, and development. IGs also often display tissue-specific expression, usually in the nervous system and testis. These characteristics translate into IG-associated diseases, mainly neuropathies, developmental disorders, and cancer. IGs represent recent additions to the genome, created mostly by retroposition of processed mRNAs with retained functionality. Processing, nuclear export, and translation of these mRNAs should be hampered dramatically by the lack of splice factors, which normally tightly cover mature transcripts and govern their fate. However, natural IGs manage to maintain satisfactory expression levels. Different mechanisms by which IGs solve the problem of mRNA processing and nuclear export are discussed here, along with their possible impact on reporter studies.

  13. Aberrant mRNA processing of the maize Rp1-D rust resistance gene in wheat and barley.

    PubMed

    Ayliffe, Michael A; Steinau, Martin; Park, Robert F; Rooke, Lee; Pacheco, Maria G; Hulbert, Scot H; Trick, Harold N; Pryor, Anthony J

    2004-08-01

    The maize Rp1-D gene confers race-specific resistance against Puccinia sorghi (common leaf rust) isolates containing a corresponding avrRp1-D avirulence gene. An Rp1-D genomic clone and a similar Rp1-D transgene regulated by the maize ubiquitin promoter were transformed independently into susceptible maize lines and shown to confer Rp1-D resistance, demonstrating that this resistance can be transferred as a single gene. Transfer of these functional transgenes into wheat and barley did not result in novel resistances when these plants were challenged with isolates of wheat stem rust (P. graminis), wheat leaf rust (P. triticina), or barley leaf rust (P. hordei). Regardless of the promoter employed, low levels of gene expression were observed. When constitutive promoters were used for transgene expression, a majority of Rp1-D transcripts were truncated in the nucleotide binding site-encoding region by premature polyadenylation. This aberrant mRNA processing was unrelated to gene function because an inactive version of the gene also generated such transcripts. These data demonstrate that resistance gene transfer between species may not be limited only by divergence of signaling effector molecules and pathogen avirulence ligands, but potentially also by more fundamental gene expression and transcript processing limitations. PMID:15305606

  14. merA gene expression in aquatic environments measured by mRNA production and Hg(II) volatilization.

    PubMed Central

    Nazaret, S; Jeffrey, W H; Saouter, E; Von Haven, R; Barkay, T

    1994-01-01

    The relationship of merA gene expression (specifying the enzyme mercuric reductase) to mercury volatilization in aquatic microbial communities was investigated with samples collected at a mercury-contaminated freshwater pond, Reality Lake, in Oak Ridge, Tenn. Levels of merA mRNA transcripts and the rate of inorganic mercury [Hg(II)] volatilization were related to the concentration of mercury in the water and to heterotrophic activity in field samples and laboratory incubations of pond water in which microbial heterotrophic activity and Hg(II) concentration were manipulated. Levels of merA-specific mRNA and Hg(II) volatilization were influenced more by microbial metabolic activity than by the concentration of mercury. merA-specific transcripts were detected in some samples which did not reduce Hg(II), suggesting that rates of mercury volatilization in environmental samples may not always be proportional to merA expression. PMID:7527625

  15. Ochratoxin a lowers mRNA levels of genes encoding for key proteins of liver cell metabolism.

    PubMed

    Hundhausen, Christoph; Boesch-Saadatmandi, Christine; Matzner, Nicole; Lang, Florian; Blank, Ralf; Wolffram, Siegfried; Blaschek, Wolfgang; Rimbach, Gerald

    2008-01-01

    Ochratoxin A (OTA) is a nephro- and hepatotoxic mycotoxin that frequently contaminates food and feedstuffs. Although recent studies have indicated that OTA modulates renal gene expression, little is known regarding its impact on differential gene expression in the liver. Therefore a microarray study of the HepG2 liver cell transcriptome in response to OTA exposure (0, 0.25, 2.5 micromol/l for 24 h) was performed using Affymetrix GeneChip technology. Selected microarray results were verified by real-time PCR and Western blotting as independent methods. Out of 14,500 genes present on the microarray, 13 and 250 genes were down-regulated by 0.25 and 2.5 micromol/l OTA, respectively. Reduced mRNA levels of calcineurin A beta (PPP3CB), which regulates inflammatory signalling pathways in immune cells, and of the uncoupling protein 2 (UCP2), which has been suggested to control the production of reactive oxygen species (ROS), were observed in response to 0.25 micromol/l OTA. A particularly strong down-regulation due to 2.5 micromol/l OTA was evident for the mRNA levels of insulin-like growth factor binding protein 1 (IGFBP1) and tubulin beta 1 (TUBB1) which have been demonstrated to function as a pro-survival factor in hepatocytes and as an important cytoskeletal component, respectively. In addition, many genes involved in energy and xenobiotic metabolism, including phosphoglycerate kinase 1 (PGK1), stearoyl-Coenzyme A desaturase 1 (SCD), and glutathione S-transferase omega 1 (GSTO1), were down-regulated by OTA. Furthermore, OTA significantly inhibited the capacitative calcium entry into the HepG2 cells, indicating an alteration of calcium homeostasis. Overall, OTA dose-dependently affects multiple genes encoding for key proteins of liver cell metabolism. PMID:19287073

  16. Depletion of three combined THOC5 mRNA export protein target genes synergistically induces human hepatocellular carcinoma cell death.

    PubMed

    Saran, S; Tran, D D H; Ewald, F; Koch, A; Hoffmann, A; Koch, M; Nashan, B; Tamura, T

    2016-07-21

    Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy. PMID:26549021

  17. Profiles of mRNA expression for genes involved in implantation, early and middle phases of secretion stage in human endometrium.

    PubMed

    Maslova, M A; Smol'nikova, V Yu; Savilova, A M; Burmenskaya, O V; Bystritskii, A A; Tabolova, V K; Korneeva, I E; Demura, T A; Donnikov, A E

    2015-04-01

    The expression of mRNA of 36 genes involved in implantation was studied by reverse transcription and real-time PCR. Significant differences in mRNA expression during the early and middle stages of the secretion phase were detected for genes mmp7, vegf, il2m, il1β, il8, il18, tnfα, il10, tgfβ, igfbp2, etc. PMID:25894777

  18. Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

    SciTech Connect

    Maquat, Lynne

    2002-12-01

    The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryotic nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?

  19. Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis.

    PubMed

    Li, Peng; Zha, Jie; Zhang, Zhenhua; Huang, Hua; Sun, Hongying; Song, Daxiang; Zhou, Kaiya

    2009-07-01

    HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships. PMID:19166961

  20. p53 Pulses Diversify Target Gene Expression Dynamics in an mRNA Half-Life-Dependent Manner and Delineate Co-regulated Target Gene Subnetworks.

    PubMed

    Porter, Joshua R; Fisher, Brian E; Batchelor, Eric

    2016-04-27

    The transcription factor p53 responds to DNA double-strand breaks by increasing in concentration in a series of pulses of fixed amplitude, duration, and period. How p53 pulses influence the dynamics of p53 target gene expression is not understood. Here, we show that, in bulk cell populations, patterns of p53 target gene expression cluster into groups with stereotyped temporal behaviors, including pulsing and rising dynamics. These behaviors correlate statistically with the mRNA decay rates of target genes: short mRNA half-lives produce pulses of gene expression. This relationship can be recapitulated by mathematical models of p53-dependent gene expression in single cells and cell populations. Single-cell transcriptional profiling demonstrates that expression of a subset of p53 target genes is coordinated across time within single cells; p53 pulsing attenuates this coordination. These results help delineate how p53 orchestrates the complex DNA damage response and give insight into the function of pulsatile signaling pathways. PMID:27135539

  1. The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing.

    PubMed Central

    Hodges, P E; Rosenberg, L E

    1989-01-01

    Ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is a mitochondrial matrix enzyme of the mammalian urea cycle. The X chromosome-linked spfash mutation in the mouse causes partial ornithine transcarbamylase deficiency and has served as a model for the human disease. We show here that the spfash mutation is a guanine to adenine transition of the last nucleotide of the fourth exon of the ornithine transcarbamylase gene. This nucleotide change produces two remarkably different effects. First, this transition causes ornithine transcarbamylase mRNA deficiency because the involved exon nucleotide plays a part in the recognition of the adjacent splice donor site. As a result of the mutation, ornithine transcarbamylase pre-mRNA is spliced inefficiently both at this site and at a cryptic splice donor site 48 bases into the adjacent intron. Second, two mutant proteins are translated from these mRNAs. From the correctly spliced mRNA, the transition results in a change of amino acid 129 from arginine to histidine. This missense substitution has no discernable effect on mitochondrial import, subunit assembly, or enzyme activity. On the other hand, the elongated mRNA resulting from mis-splicing is translated into a dysfunctional ornithine transcarbamylase subunit elongated by the insertion of 16 amino acid residues. Images PMID:2471197

  2. Quantification of diatom gene expression in the sea by selecting uniformly transcribed mRNA as the basis for normalization.

    PubMed

    Kang, Lee-Kuo; Tsui, Feng-Hsiu; Chang, Jeng

    2012-09-01

    To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene. PMID:22706063

  3. Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

    PubMed Central

    Kang, Lee-Kuo; Tsui, Feng-Hsiu

    2012-01-01

    To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene. PMID:22706063

  4. Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

    PubMed

    Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

    2016-04-15

    Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. PMID:26787513

  5. Influence of beta-blockers on the myocardial mRNA expressions of circadian clock- and metabolism-related genes.

    PubMed

    Ushijima, Kentarou; Maekawa, Tomohiro; Ishikawa-Kobayashi, Eiko; Ando, Hitoshi; Shiga, Tsuyoshi; Fujimura, Akio

    2013-01-01

    Daily rhythms are regulated by a master clock-system in the suprachiasmatic nucleus and by a peripheral clock-system in each organ. Because norepinephrine is one of the timekeepers for the myocardial circadian clock that influences cardiac metabolism, it is speculated that a beta-blocker may affect the circadian clock and metabolism in heart tissue. In this study, thirty mg/kg/day of propranolol (a lipophilic beta-blocker) or atenolol (a hydrophilic beta-blocker) was given orally to Wistar rats for 4 weeks. The mRNA expressions of Bmal1 and E4BP4 in heart tissue were suppressed by the beta-blockers. However, the mRNA expressions of these clock genes in the suprachiasmatic nucleus were unchanged. Myocardial mRNA expressions of lactate dehydrogenase a and pyruvate dehydrogenase kinase 4 were also suppressed by the beta-blockers. In addition, ATP content in heart tissue was significantly elevated by the beta-blockers throughout 24 hours. The effects of propranolol and atenolol did not differ significantly. This study showed for the first time that a beta-blocker affects myocardial clock gene expression. Propranolol and atenolol increased ATP content in heart tissue throughout 24 hours. The influences of beta-blockers may be negligible on the SCN, and may be independent of lipid solubility on heart tissue. It is well known that these drugs exert a protective effect against myocardial ischemia, which may be mediated by an increase in the preservation of myocardial ATP. PMID:23394803

  6. Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

    NASA Technical Reports Server (NTRS)

    Shibata, K.; Abe, S.; Davies, E.

    2001-01-01

    Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

  7. Gene (mRNA) expression in canine atopic dermatitis: microarray analysis.

    PubMed

    Merryman-Simpson, Annemarie E; Wood, Shona H; Fretwell, Neale; Jones, Paul G; McLaren, William M; McEwan, Neil A; Clements, Dylan N; Carter, Stuart D; Ollier, William E; Nuttall, Tim

    2008-04-01

    Genes potentially involved in the pathology of canine atopic dermatitis (AD) were identified using gene expression microarrays. Total RNA extracted from skin biopsies was hybridized to an Agilent Technologies custom-designed 22K canine array. The arrays were analysed using Genedata Analyst software. Data were corrected for multiple hypothesis testing and tested for significance using the National Institute on Aging array analysis tool. For comparison, data were divided into separate groups: lesional atopic (n = 16), nonlesional atopic (n = 17) and healthy controls (n = 9). Fifty-four genes were differentially expressed at a significance level of 0.05 in canine AD compared to healthy controls. Sixteen genes were differentially expressed in both nonlesional and lesional atopic skin, 26 genes only in nonlesional skin and 12 only in lesional skin. These genes were associated with innate immune and inflammatory responses, cell cycle, apoptosis, barrier formation and transcriptional regulation. The most dysregulated gene in lesional skin was S100A8, which showed an almost 23-fold increase in expression. This is a pro-inflammatory cytokine located in the epidermal differentiation complex. Microarray analysis is a novel technique in canine AD. Significant changes in gene expression were identified in atopic skin. These were relevant to skin barrier formation and the immune response, suggesting that they both participate in AD. Gene expression restricted to lesional skin may be involved in inflammatory changes, whereas those shared or restricted to nonlesional skin may reflect the atopic phenotype. Investigating gene polymorphisms in the targets identified in this study will help improve our understanding of the genetic basis of this disease. PMID:18336422

  8. Integrated Analysis of DNA Methylation and mRNA Expression Profiles Data to Identify Key Genes in Lung Adenocarcinoma.

    PubMed

    Jin, Xiang; Liu, Xingang; Li, Xiaodan; Guan, Yinghui

    2016-01-01

    Introduction. Lung adenocarcinoma (LAC) is the most frequent type of lung cancer and has a high metastatic rate at an early stage. This study is aimed at identifying LAC-associated genes. Materials and Methods. GSE62950 downloaded from Gene Expression Omnibus included a DNA methylation dataset and an mRNA expression profiles dataset, both of which included 28 LAC tissue samples and 28 adjacent normal tissue samples. The differentially expressed genes (DEGs) were screened by Limma package in R, and their functions were predicted by enrichment analysis using TargetMine online tool. Then, protein-protein interaction (PPI) network was constructed using STRING and Cytoscape. Finally, LAC-associated methylation sites were identified by CpGassoc package in R and mapped to the DEGs to obtain LAC-associated DEGs. Results. Total 913 DEGs were identified in LAC tissues. In the PPI networks, MAD2L1, AURKB, CCNB2, CDC20, and WNT3A had higher degrees, and the first four genes might be involved in LAC through interaction. Total 8856 LAC-associated methylation sites were identified and mapped to the DEGs. And there were 29 LAC-associated methylation sites located in 27 DEGs (e.g., SH3GL2, BAI3, CDH13, JAM2, MT1A, LHX6, and IGFBP3). Conclusions. These key genes might play a role in pathogenesis of LAC. PMID:27610375

  9. Integrated Analysis of DNA Methylation and mRNA Expression Profiles Data to Identify Key Genes in Lung Adenocarcinoma

    PubMed Central

    Jin, Xiang; Li, Xiaodan; Guan, Yinghui

    2016-01-01

    Introduction. Lung adenocarcinoma (LAC) is the most frequent type of lung cancer and has a high metastatic rate at an early stage. This study is aimed at identifying LAC-associated genes. Materials and Methods. GSE62950 downloaded from Gene Expression Omnibus included a DNA methylation dataset and an mRNA expression profiles dataset, both of which included 28 LAC tissue samples and 28 adjacent normal tissue samples. The differentially expressed genes (DEGs) were screened by Limma package in R, and their functions were predicted by enrichment analysis using TargetMine online tool. Then, protein-protein interaction (PPI) network was constructed using STRING and Cytoscape. Finally, LAC-associated methylation sites were identified by CpGassoc package in R and mapped to the DEGs to obtain LAC-associated DEGs. Results. Total 913 DEGs were identified in LAC tissues. In the PPI networks, MAD2L1, AURKB, CCNB2, CDC20, and WNT3A had higher degrees, and the first four genes might be involved in LAC through interaction. Total 8856 LAC-associated methylation sites were identified and mapped to the DEGs. And there were 29 LAC-associated methylation sites located in 27 DEGs (e.g., SH3GL2, BAI3, CDH13, JAM2, MT1A, LHX6, and IGFBP3). Conclusions. These key genes might play a role in pathogenesis of LAC. PMID:27610375

  10. Exploiting aberrant mRNA expression in autism for gene discovery and diagnosis.

    PubMed

    Guan, Jinting; Yang, Ence; Yang, Jizhou; Zeng, Yong; Ji, Guoli; Cai, James J

    2016-07-01

    Autism spectrum disorder (ASD) is characterized by substantial phenotypic and genetic heterogeneity, which greatly complicates the identification of genetic factors that contribute to the disease. Study designs have mainly focused on group differences between cases and controls. The problem is that, by their nature, group difference-based methods (e.g., differential expression analysis) blur or collapse the heterogeneity within groups. By ignoring genes with variable within-group expression, an important axis of genetic heterogeneity contributing to expression variability among affected individuals has been overlooked. To this end, we develop a new gene expression analysis method-aberrant gene expression analysis, based on the multivariate distance commonly used for outlier detection. Our method detects the discrepancies in gene expression dispersion between groups and identifies genes with significantly different expression variability. Using this new method, we re-visited RNA sequencing data generated from post-mortem brain tissues of 47 ASD and 57 control samples. We identified 54 functional gene sets whose expression dispersion in ASD samples is more pronounced than that in controls, as well as 76 co-expression modules present in controls but absent in ASD samples due to ASD-specific aberrant gene expression. We also exploited aberrantly expressed genes as biomarkers for ASD diagnosis. With a whole blood expression data set, we identified three aberrantly expressed gene sets whose expression levels serve as discriminating variables achieving >70 % classification accuracy. In summary, our method represents a novel discovery and diagnostic strategy for ASD. Our findings may help open an expression variability-centered research avenue for other genetically heterogeneous disorders. PMID:27131873

  11. The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in vivo

    SciTech Connect

    Carrazana, E.J.; Pasieka, K.B.; Majzoub, J.A.

    1988-06-01

    The authors developed a method, termed an H-blot, by which the poly(A) tract of any specific mRNA may be detected by RNA filter hybridization after its removal from the body of the mRNA by a RNase H-catalyzed endonucleolytic cleavage in the 3' untranslated region. Using this method, they studied the modulation of the length of the poly(A) tract of rat vasopressin mRNA in vivo during changes in the levels of this mRNA resulting from a physiologic stimulus, osmotic stress. The poly(A) tract of hypothalamic vasopressin mRNA in hydrated rats was, quite remarkably, --250 nucleotides in length, in contrast to that of somatostatin mRNA, which was --30 nucleotides long. Vasopressin mRNA poly(A) tail length increased progressively from --250 to --400 nucleotides with the application of the hyperosmotic stimulus and declined to base line after its removal; somatostatin mRNA poly(A) tail length did not change during osmotic stress. The poly(A) tract length of total hypothalamic mRNA was between 35 and 140 nucleotides and also did not change with osmotic stress. Modulation of poly(A) tract length of specific mRNAs during stimulation of gene expression may provide an additional level of genetic regulation.

  12. The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in vivo.

    PubMed Central

    Carrazana, E J; Pasieka, K B; Majzoub, J A

    1988-01-01

    We developed a method, termed an H-blot, by which the poly(A) tract of any specific mRNA may be detected by RNA filter hybridization after its removal from the body of the mRNA by a RNase H-catalyzed endonucleolytic cleavage in the 3' untranslated region. Using this method, we studied the modulation of the length of the poly(A) tract of rat vasopressin mRNA in vivo during changes in the levels of this mRNA resulting from a physiologic stimulus, osmotic stress. The poly(A) tract of hypothalamic vasopressin mRNA in hydrated rats was, quite remarkably, approximately 250 nucleotides in length, in contrast to that of somatostatin mRNA, which was approximately 30 nucleotides long. Vasopressin mRNA poly(A) tail length increased progressively from approximately 250 to approximately 400 nucleotides with the application of the hyperosmotic stimulus and declined to base line after its removal; somatostatin mRNA poly(A) tail length did not change during osmotic stress. The poly(A) tract length of total hypothalamic mRNA was between 35 and 140 nucleotides and also did not change with osmotic stress. Modulation of poly(A) tract length of specific mRNAs during stimulation of gene expression may provide an additional level of genetic regulation. Images PMID:2841576

  13. Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence

    PubMed Central

    Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; D. van der Vaart, Andrew; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S.; Miles, Michael F.; Dick, Danielle; Riley, Brien P.; Dumur, Catherine; Vladimirov, Vladimir I.

    2015-01-01

    Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

  14. Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence.

    PubMed

    Mamdani, Mohammed; Williamson, Vernell; McMichael, Gowon O; Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; van der Vaart, Andrew D; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S; Miles, Michael F; Dick, Danielle; Riley, Brien P; Dumur, Catherine; Vladimirov, Vladimir I

    2015-01-01

    Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

  15. Prenylation differentially inhibits insulin-dependent immediate early gene mRNA expression.

    PubMed

    Franklin, J Lee; Amsler, Maggie O; Messina, Joseph L

    2016-06-01

    Increased activity of prenyl transferases is observed in pathological states of insulin resistance, diabetes, and obesity. Thus, functional inhibitors of farnesyl transferase (FTase) and geranylgeranyl transferase (GGTase) may be promising therapeutic treatments. We previously identified insulin responsive genes from a rat H4IIE hepatoma cell cDNA library, including β-actin, EGR1, Pip92, c-fos, and Hsp60. In the present study, we investigated whether acute treatment with FTase and GGTase inhibitors would alter insulin responsive gene initiation and/or elongation rates. We observed differential regulation of insulin responsive gene expression, suggesting a differential sensitivity of these genes to one or both of the specific protein prenylation inhibitors. PMID:27086854

  16. Polymorphisms of the IL8 gene correlate with milking traits, SCS and mRNA level in Chinese Holstein.

    PubMed

    Chen, Renjin; Yang, Zhangping; Ji, Dejun; Mao, Yongjiang; Chen, Ying; Li, Yunlong; Wu, Haitao; Wang, Xiaolong; Chang, Lingling

    2011-08-01

    To explore the relation of Interleukin-8 (IL8) gene polymorphism with immunity against mastitis in dairy cow, the polymorphism of IL8 gene was investigated in 610 Chinese Holstein cow from 30 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, and milk yield, milk fat percentage, milk protein percentage, 305 day corrected milk yield, 305 day milk fat yield, 305 day milk protein yield and somatic cell score (SCS) were measured and analyzed, and the mRNA levels of IL8 genotypes in blood were detected by real-time PCR. The results showed that three genotypes, KK, KA and AA were detected with frequencies of 0.187, 0.451, and 0.362, respectively. The gene frequencies of K and A were 0.412 and 0.588, respectively. The significant association of the IL8 mutations with milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield, SCS, and significant association with milk protein percentage were identified, while their association with milk fat percentage were not significant. KK had higher milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield than AA or KA, and the least square mean of SCS of KK was significantly lower than that of AA or KA. AA had significant lower milk protein yield than KK or KA. The relative IL8 mRNA level of KK in blood was the highest. These findings demonstrated that IL8 genotype significantly correlated with mastitis resistance and the locus could be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein. PMID:21113675

  17. Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

    PubMed Central

    Garrett, J E; Collard, M W; Douglass, J O

    1989-01-01

    The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed. Images PMID:2573832

  18. Cholesterol Status Modulates mRNA and Protein Levels of Genes Associated with Cholesterol Metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary saturated (S), monounsaturated (MU) and polyunsaturated (PU) fatty acids (FA) and cholesterol have been shown to be major determinants of plasma lipoprotein profiles. The objective was to determine the effect of whole body cholesterol status and dietary fatty acid saturation on genes associ...

  19. Control of gene expression by mRNA transport and turnover

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes are regulated primarily by their rate of transcript initiation. However, recent research in eukaryotic transcript degradation, export, and localization indicate that these processes contribute significantly to the amount of any given transcript in a cell. This review chapter looks at general...

  20. Early gene expression in bacteriophage T7. I. In vivo synthesis, inactivation, and translational utilization of early mRNA's.

    PubMed

    Hercules, K; Jovanovich, S; Sauerbrier, W

    1976-02-01

    In vivo decay rates for the individual T7 early mRNA species were determined. The physical half-lives, measured at 37 C, range from 1.1 min for gene 0.7 RNA to 4.5 min for gene 0.3 RNA. Physical half-lives, as observed after rifampin inhibition of RNA synthesis and polyacylamide electrophoresis of RNAs, are approximately 30% longer than functional half-lives, as observed by 14C-labeled amino acid uptake into individual T7 early proteins. The different RNA species are synthesized at grossly different rates, 0.3 RNA at four times the rate of 1.0 RNA, 0.7 RNA at twice the rate, and 1.1 and 1.3 RNAs at about the same or a slightly lower rate than 1.0 RNA. Rho-factor-mediated termination of transcription behind genes 0.3, 0.7, and perhaps behind 1.0 is inferred from these data. The in vivo translational utilization of the individual T7 early-message species was found to vary by not more than a factor of 2. PMID:1255850

  1. Parameter estimation methods for gene circuit modeling from time-series mRNA data: a comparative study.

    PubMed

    Fan, Ming; Kuwahara, Hiroyuki; Wang, Xiaolei; Wang, Suojin; Gao, Xin

    2015-11-01

    Parameter estimation is a challenging computational problem in the reverse engineering of biological systems. Because advances in biotechnology have facilitated wide availability of time-series gene expression data, systematic parameter estimation of gene circuit models from such time-series mRNA data has become an important method for quantitatively dissecting the regulation of gene expression. By focusing on the modeling of gene circuits, we examine here the performance of three types of state-of-the-art parameter estimation methods: population-based methods, online methods and model-decomposition-based methods. Our results show that certain population-based methods are able to generate high-quality parameter solutions. The performance of these methods, however, is heavily dependent on the size of the parameter search space, and their computational requirements substantially increase as the size of the search space increases. In comparison, online methods and model decomposition-based methods are computationally faster alternatives and are less dependent on the size of the search space. Among other things, our results show that a hybrid approach that augments computationally fast methods with local search as a subsequent refinement procedure can substantially increase the quality of their parameter estimates to the level on par with the best solution obtained from the population-based methods while maintaining high computational speed. These suggest that such hybrid methods can be a promising alternative to the more commonly used population-based methods for parameter estimation of gene circuit models when limited prior knowledge about the underlying regulatory mechanisms makes the size of the parameter search space vastly large. PMID:25818863

  2. An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

    PubMed

    Devonshire, Alison S; Sanders, Rebecca; Whale, Alexandra S; Nixon, Gavin J; Cowen, Simon; Ellison, Stephen L R; Parkes, Helen; Pine, P Scott; Salit, Marc; McDaniel, Jennifer; Munro, Sarah; Lund, Steve; Matsukura, Satoko; Sekiguchi, Yuji; Kawaharasaki, Mamoru; Granjeiro, José Mauro; Falagan-Lotsch, Priscila; Saraiva, Antonio Marcos; Couto, Paulo; Yang, Inchul; Kwon, Hyerim; Park, Sang-Ryoul; Demšar, Tina; Žel, Jana; Blejec, Andrej; Milavec, Mojca; Dong, Lianhua; Zhang, Ling; Sui, Zhiwei; Wang, Jing; Viroonudomphol, Duangkamol; Prawettongsopon, Chaiwat; Partis, Lina; Baoutina, Anna; Emslie, Kerry; Takatsu, Akiko; Akyurek, Sema; Akgoz, Muslum; Vonsky, Maxim; Konopelko, L A; Cundapi, Edna Matus; Urquiza, Melina Pérez; Huggett, Jim F; Foy, Carole A

    2016-06-01

    Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the

  3. Estrogen-dependent activation of the avian very low density apolipoprotein II and vitellogenin genes. Transient alterations in mRNA polyadenylation and stability early during induction.

    PubMed

    Cochrane, A W; Deeley, R G

    1988-10-01

    Administration of estrogen to egg-laying vertebrates activates unscheduled, hepatic expression of major, egg-yolk protein genes in immature animals and mature males. Two avian yolk protein genes, encoding very low density apolipoprotein II (apoVLDLII) and vitellogenin II, are dormant prior to stimulation with estrogen, but within three days their cognate mRNAs accumulate to become two of the most abundant species in the liver. Accumulation of these mRNAs has been attributed to both induction of transcription and selective, estrogen-dependent mRNA stabilization. We have detected alterations in the size of apoVLDLII mRNA that occur during the first 24 hours that are attributable to a shift in the extent of polyadenylation as steady-state is approached. In vitro transcription assays indicate that primary activation of both genes takes place relatively slowly and that maximal rates of mRNA accumulation occur when the apoVLDLII and vitellogenin II genes are expressed at only 30% and 10% of their fully induced levels, respectively. Transcription data combined with the structural alteration of apoVLDLII mRNA suggest that stability of the two mRNAs may change as steady-state is approached. We have assessed the compatibility of this suggestion with earlier estimates of the kinetics of accumulation of both mRNAs by developing a generally useful algorithm that predicts approach to steady-state kinetics under conditions where both the rate of synthesis and mRNA stability change throughout the accumulation phase of the response. The results predict that the stability of both mRNAs decreases by at least two- to threefold during the approach to steady-state and that, although an additional destabilization of apoVLDLII mRNA may occur following withdrawal of estrogen, the steady-state stability of vitellogenin mRNA is not significantly decreased upon removal of hormone. PMID:3210227

  4. Comparative analyses of gene copy number and mRNA expression in GBM tumors and GBM xenografts

    SciTech Connect

    Hodgson, J. Graeme; Yeh, Ru-Fang; Ray, Amrita; Wang, Nicholas J.; Smirnov, Ivan; Yu, Mamie; Hariono, Sujatmi; Silber, Joachim; Feiler, Heidi S.; Gray, Joe W.; Spellman, Paul T.; Vandenberg, Scott R.; Berger, Mitchel S.; James, C. David

    2009-04-03

    Development of model systems that recapitulate the molecular heterogeneity observed among glioblastoma multiforme (GBM) tumors will expedite the testing of targeted molecular therapeutic strategies for GBM treatment. In this study, we profiled DNA copy number and mRNA expression in 21 independent GBM tumor lines maintained as subcutaneous xenografts (GBMX), and compared GBMX molecular signatures to those observed in GBM clinical specimens derived from the Cancer Genome Atlas (TCGA). The predominant copy number signature in both tumor groups was defined by chromosome-7 gain/chromosome-10 loss, a poor-prognosis genetic signature. We also observed, at frequencies similar to that detected in TCGA GBM tumors, genomic amplification and overexpression of known GBM oncogenes, such as EGFR, MDM2, CDK6, and MYCN, and novel genes, including NUP107, SLC35E3, MMP1, MMP13, and DDX1. The transcriptional signature of GBMX tumors, which was stable over multiple subcutaneous passages, was defined by overexpression of genes involved in M phase, DNA replication, and chromosome organization (MRC) and was highly similar to the poor-prognosis mitosis and cell-cycle module (MCM) in GBM. Assessment of gene expression in TCGA-derived GBMs revealed overexpression of MRC cancer genes AURKB, BIRC5, CCNB1, CCNB2, CDC2, CDK2, and FOXM1, which form a transcriptional network important for G2/M progression and/or checkpoint activation. Our study supports propagation of GBM tumors as subcutaneous xenografts as a useful approach for sustaining key molecular characteristics of patient tumors, and highlights therapeutic opportunities conferred by this GBMX tumor panel for testing targeted therapeutic strategies for GBM treatment.

  5. Colorimetric In Situ Hybridization Identifies MYC Gene Signal Clusters Correlating With Increased Copy Number, mRNA, and Protein in Diffuse Large B-cell Lymphoma

    PubMed Central

    Valentino, Carlo; Kendrick, Samantha; Johnson, Nathalie; Gascoyne, Randy; Chan, Wing C.; Weisenburger, Dennis; Braziel, Rita; Cook, James R.; Tubbs, Raymond; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Jaffe, Elaine; Zhang, Wenjun; Brunhoeber, Patrick; Nitta, Hiro; Grogan, Tom; Rimsza, Lisa

    2014-01-01

    Abnormalities of the MYC oncogene on chromosome 8 are characteristic of Burkitt lymphoma and other aggressive B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL). We recently described a colorimetric in situ hybridization (CISH) method for detecting extra copies of the MYC gene in DLBCL and the frequent occurrence of excess copies of discrete MYC signals in the context of diploidy or polyploidy of chromosome 8, which correlated with increased mRNA signals. We further observed enlarged MYC signals, which were counted as a single gene copy but, by their dimension and unusual shape, likely consisted of “clusters” of MYC genes. In this study, we sought to further characterize these clusters of MYC signals by determining whether the presence of these correlated with other genetic features, mRNA levels, protein, and overall survival. We found that MYC clusters correlated with an abnormal MYC locus and with increased mRNA. MYC mRNA correlated with protein levels, and both increased mRNA and protein correlated with poorer overall survival. MYC clusters were seen in both the germinal center and activated B-cell subtypes of DLBCL. Clusters of MYC signals may be an underappreciated, but clinically important, feature of aggressive B-cell lymphomas with potential prognostic and therapeutic relevance. PMID:23355209

  6. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR.

    PubMed

    DeLoughery, Aaron; Dengler, Vanina; Chai, Yunrong; Losick, Richard

    2016-01-01

    Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We also show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex. PMID:26434553

  7. In situ, real-time catabolic gene expression: Extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater

    SciTech Connect

    Wilson, M.S.; Bakermans, C.; Madsen, E.L.

    1999-01-01

    The authors developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-{micro}m-pore-size filters, which were then frozen to dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs. Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To the authors` knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.

  8. Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3′ ends

    PubMed Central

    Liu, Bo; Kearns, Daniel B.; Bechhofer, David H.

    2016-01-01

    Timely turnover of RNA is an important element in the control of bacterial gene expression, but relatively few specific targets of RNA turnover regulation are known. Deletion of the Bacillus subtilis pnpA gene, encoding the major 3′ exonuclease turnover enzyme, polynucleotide phosphorylase (PNPase), was shown previously to cause a motility defect correlated with a reduced level of the 32-gene fla/che flagellar biosynthesis operon transcript. fla/che operon transcript abundance has been shown to be inhibited by an excess of the small regulatory protein, SlrA, and here we find that slrA mRNA accumulated in the pnpA-deletion mutant. Mutation of slrA was epistatic to mutation of pnpA for the motility-related phenotype. Further, Rho-dependent termination was required for PNPase turnover of slrA mRNA. When the slrA gene was provided with a Rho-independent transcription terminator, gene regulation was no longer PNPase-dependent. Thus we show that the slrA transcript is a direct target of PNPase and that regulation of RNA turnover is a major determinant of motility gene expression. The interplay of specific transcription termination and mRNA decay mechanisms suggests selection for fine-tuning of gene expression. PMID:26857544

  9. Methylation and mRNA expression levels of P15, death-associated protein kinase, and suppressor of cytokine signaling-1 genes in multiple myeloma

    PubMed Central

    Liu, Lin; Tan, Lin; He, Zhenxin

    2016-01-01

    Objective(s): The aim of this study was to investigate the methylation status and mRNA expression levels of P15, death-associated protein kinase (DAPK), and suppressor of cytokine signaling-1 (SOCS1) genes in multiple myeloma (MM). Materials and Methods: The bone marrow samples of 54 MM patients were collected and the methylation status of the P15, DAPK, and SOCS1 gene promoter regions was determined by methylation-specific polymerase chain reaction. Automated sequencing technology was used to sequence the amplified products in order to analyze the base methylation sites. mRNA expression levels were determined using real-time fluorescent quantitative polymerase chain reaction. Results: Among the 54 MM patients, the positive methylation rates of the P15, DAPK, and SOCS1 genes were 27.78%, 18.52%, and 16.67%, respectively. The methylation results were confirmed by sequencing. The positive methylation rates of the P15, DAPK, and SOCS1 genes showed no correlation with patient gender, age, typing, staging, and grouping (P>0.05). There was no significant difference in the mRNA expression levels of the P15, DAPK, and SOCS1 genes between the MM patient group and the control group (P>0.05). Conclusions: Aberrant methylation of the P15, DAPK, and SOCS1 genes exists in MM, and these genes may play certain roles in pathogenesis of MM. There was no significant difference in mRNA expression levels between the methylated group and the non-methylated group, suggesting that these genes are regulated by other mechanisms during their transcription.

  10. Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics

    PubMed Central

    Wei, Youheng; Xiao, Qianghai; Zhang, Ting; Mou, Zongchun; You, Jia; Ma, Wei-Jun

    2009-01-01

    The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3′-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly. PMID:19726583

  11. Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics.

    PubMed

    Wei, Youheng; Xiao, Qianghai; Zhang, Ting; Mou, Zongchun; You, Jia; Ma, Wei-Jun

    2009-10-01

    The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3'-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly. PMID:19726583

  12. Changes in Denitrifier Abundance, Denitrification Gene mRNA Levels, Nitrous Oxide Emissions, and Denitrification in Anoxic Soil Microcosms Amended with Glucose and Plant Residues▿

    PubMed Central

    Henderson, Sherri L.; Dandie, Catherine E.; Patten, Cheryl L.; Zebarth, Bernie J.; Burton, David L.; Trevors, Jack T.; Goyer, Claudia

    2010-01-01

    In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification. The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene mRNA levels, nitrous oxide (N2O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h. nosZ gene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast, the abundance and mRNA levels of Pseudomonas mandelii and closely related species (nirSP) increased only in glucose-amended soil: the nirSP guild abundance increased 5-fold over the 72-h incubation period (P < 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P < 0.001) and then subsequently decreased. The nosZ gene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen (C:N) ratios varied from 15:1 to 30:1, nosZ gene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 × 107 gene copies and 6.9 × 107 transcripts g−1 dry soil. Cumulative N2O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirSP and nosZ communities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic C and different C:N ratios. PMID:20154105

  13. Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver

    SciTech Connect

    Houldsworth, J.; Attardi, G.

    1988-01-01

    Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH/sub 2/-end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones.

  14. Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System

    PubMed Central

    Bire, Solenne; Ley, Déborah; Casteret, Sophie; Mermod, Nicolas; Bigot, Yves; Rouleux-Bonnin, Florence

    2013-01-01

    Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors. PMID:24312663

  15. FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity Across All Lung Cancer Histologies

    PubMed Central

    Wynes, Murry W.; Hinz, Trista K.; Gao, Dexiang; Martini, Michael; Marek, Lindsay A.; Ware, Kathryn E.; Edwards, Michael G.; Böhm, Diana; Perner, Sven; Helfrich, Barbara A.; Dziadziuszko, Rafal; Jassem, Jacek; Wojtylak, Szymon; Sejda, Aleksandra; Gozgit, Joseph M.; Bunn, Paul A.; Camidge, D. Ross; Tan, Aik-Choon; Hirsch, Fred R.; Heasley, Lynn E.

    2014-01-01

    Purpose FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous-cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically-relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray comprised of resected lung tumors was submitted to FGFR1 GCN and mRNA analyses and the results were validated with TCGA lung cancer data. Results 14/58 cell lines exhibited ponatinib sensitivity (IC50 values ≤ 50 nM) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions FGFR1 dependency is frequent across various lung cancer histologies and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. PMID:24771645

  16. Cholesterol Side-Chain Cleavage Gene Expression in Theca Cells: Augmented Transcriptional Regulation and mRNA Stability in Polycystic Ovary Syndrome

    PubMed Central

    Nelson-DeGrave, Velen L.; Legro, Richard S.; Strauss, Jerome F.; McAllister, Jan M.

    2012-01-01

    Hyperandrogenism is characteristic of women with polycystic ovary syndrome (PCOS). Ovarian theca cells isolated from PCOS follicles and maintained in long-term culture produce elevated levels of progestins and androgens compared to normal theca cells. Augmented steroid production in PCOS theca cells is associated with changes in the expression of genes for several steroidogenic enzymes, including CYP11A1, which encodes cytochrome P450 cholesterol side-chain cleavage. Here, we further examined CYP11A1 gene expression, at both the transcriptional and post-transcriptional level in normal and PCOS theca cells propagated in long-term culture utilizing quantitative RT-PCR, functional promoter analyses, and mRNA degradation studies. The minimal element(s) that conferred increased basal and cAMP-dependent CYP11A1 promoter function were determined. CYP11A1 mRNA half-life in normal and PCOS theca cells was compared. Results of these cumulative studies showed that basal and forskolin stimulated steady state CYP11A1 mRNA abundance and CYP11A1 promoter activity were increased in PCOS theca cells. Deletion analysis of the CYP11A1 promoter demonstrated that augmented promoter function in PCOS theca cells results from increased basal regulation conferred by a minimal sequence between −160 and −90 bp of the transcriptional start site. The transcription factor, nuclear factor 1C2, was observed to regulate basal activity of this minimal CYP11A1 element. Examination of mRNA stability in normal and PCOS theca cells demonstrated that CYP11A1 mRNA half-life increased >2-fold, from approximately 9.22+/−1.62 h in normal cells, to 22.38+/−0.92 h in PCOS cells. Forskolin treatment did not prolong CYP11A1 mRNA stability in either normal or PCOS theca cells. The 5′-UTR of CYP11A1 mRNA confers increased basal mRNA stability in PCOS cells. In conclusion, these studies show that elevated steady state CYP11A1 mRNA abundance in PCOS cells results from increased transactivation of the CYP

  17. No change in mRNA expression of immune-related genes in peripheral blood mononuclear cells challenged with Theileria annulata in Murrah buffalo (Bubalus bubalis).

    PubMed

    Panigrahi, Manjit; Kumar, Amod; Bhushan, Bharat; Ghosh, Srikant; Saravanan, B C; Sulabh, Sourabh; Parida, Subhashree; Gaur, Gyanendra Kumar

    2016-07-01

    Water buffaloes (Bubalus bubalis) act as carrier to Theileria annulata and show less clinical sign of tropical theileriosis as compared to indigenous and exotic cattle. Differential expression of immune-related genes such as major histocompatibility complex, class II, DQ alpha 1 (MHC-DQα), signal-regulatory protein alpha (SIRPA), prion protein (PRNP), Toll-like receptor 10 (TLR10), c-musculoaponeurotic fibrosarcoma oncogene homolog (cMAF) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) genes influence host resistance to this disease in exotic, crossbred and indigenous cattle. In the present study we examined the differential mRNA expression of the abovesaid immune-related genes in response to T. annulata infection in buffaloes. Peripheral blood mononuclear cells (PBMCs) harvested from blood samples of buffaloes were challenged with ground-up tick supernatant carrying T. annulata sporozoites in vitro. After 48h of in vitro challenge qPCR was employed to measure the relative mRNA expression of MHC-DQα, SIRPA, PRNP, TLR10, cMAF and MAFB genes in infected and control PBMCs. In the current study, the selected genes showed no change in mRNA expression after T.annulata infection which indicates that they have little role in providing host resistance to theileriosis in buffaloes. PMID:26997138

  18. Intrahepatic mRNA Expression of FAS, FASL, and FOXP3 Genes Is Associated with the Pathophysiology of Chronic HCV Infection

    PubMed Central

    Amoras, Ednelza da Silva Graça; Gomes, Samara Tatielle Monteiro; Freitas, Felipe Bonfim; Santana, Bárbara Brasil; Ishak, Geraldo; Ferreira de Araújo, Marialva Tereza; Demachki, Sâmia; Conde, Simone Regina Souza da Silva; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo; Vallinoto, Antonio Carlos Rosário

    2016-01-01

    This study aimed to evaluate the relative mRNA expression of Fas receptor (FAS), Fas ligand (FASL), and forkhead box protein 3 (FOXP3) in liver biopsy specimens obtained from patients with viral and non-viral chronic hepatitis and correlate their expression with the fibrosis stage. A total of 51 liver biopsy specimens obtained from HBV (n = 6), HCV (n = 28), and non-viral hepatic disease (NVHD) (n = 9) patients and from individuals with normal liver histology (n = 8) (control—CT) were analyzed. Quantifications of the target genes were assessed using qPCR, and liver biopsies according to the METAVIR classification. The mRNA expression levels of FAS and FASL were lower in the CT group compared to the groups of patients. The increase in the mRNA expression of FAS and FASL was correlated with higher levels of inflammation and disease progression, followed by a decline in tissues with cirrhosis, and it was also associated with increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Higher mRNA expression of FOXP3 was observed in the HCV and NVHD groups, with the peak observed among patients with cirrhosis. The increased FOXP3 mRNA expression was positively correlated with increased FAS and FASL mRNA expression and the AST and ALT levels in all patients. Conclusions: These results suggest that regardless of the cause, the course of chronic liver disease may be modulated by the analyzed genes and correlated with an increase in regulatory T cells during the liver damage followed by hepatocyte destruction by Fas/FasL system and subsequent non specific lymphocytic infiltrate accumulation. PMID:27243827

  19. Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing*♦

    PubMed Central

    Nageshan, Rishi Kumar; Roy, Nainita; Hehl, Adrian B.; Tatu, Utpal

    2011-01-01

    Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 (glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the “intron” regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing. PMID:21209094

  20. Post-transcriptional repair of a split heat shock protein 90 gene by mRNA trans-splicing.

    PubMed

    Nageshan, Rishi Kumar; Roy, Nainita; Hehl, Adrian B; Tatu, Utpal

    2011-03-01

    Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 (glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the "intron" regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing. PMID:21209094

  1. In Vivo mRNA Profiling of Uropathogenic Escherichia coli from Diverse Phylogroups Reveals Common and Group-Specific Gene Expression Profiles

    PubMed Central

    Bielecki, Piotr; Muthukumarasamy, Uthayakumar; Eckweiler, Denitsa; Bielecka, Agata; Pohl, Sarah; Schanz, Ansgar; Niemeyer, Ute; Oumeraci, Tonio; von Neuhoff, Nils; Ghigo, Jean-Marc

    2014-01-01

    ABSTRACT mRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression of Escherichia coli pathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associated E. coli isolates to different phylogenetic groups. Whereas the in vivo gene expression profiles of the majority of genes were conserved among 21 E. coli strains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribed in vivo relative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease. PMID:25096872

  2. The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA.

    PubMed Central

    Boot, H J; Kolen, C P; Andreadaki, F J; Leer, R J; Pouwels, P H

    1996-01-01

    S-proteins are proteins which form a regular structure (S-layer) on the outside of the cell walls of many bacteria. Two S-protein-encoding genes are located in opposite directions on a 6.0-kb segment of the chromosome of Lactobacillus acidophilus ATCC 4356 bacteria. Inversion of this chromosomal segment occurs through recombination between two regions with identical sequences, thereby interchanging the expressed and the silent genes. In this study, we show that the region involved in recombination also has a function in efficient S-protein production. Two promoter sequences are present in the S-protein gene expression site, although only the most downstream promoter (P-1) is used to direct mRNA synthesis. S-protein mRNA directed by this promoter has a half-life of 15 min. Its untranslated leader can form a stable secondary structure in which the 5' end is base paired, whereas the ribosome-binding site is exposed. Truncation of this leader sequence results in a reduction in protein production, as shown by reporter gene analysis of Lactobacillus casei. The results obtained indicate that the untranslated leader sequence of S-protein mRNA is involved in efficient S-protein production. PMID:8808926

  3. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 Is Required for Circadian Periodicity through the Promotion of Nucleo-Cytoplasmic mRNA Export in Arabidopsis[W][OPEN

    PubMed Central

    MacGregor, Dana R.; Gould, Peter; Foreman, Julia; Griffiths, Jayne; Bird, Susannah; Page, Rhiannon; Stewart, Kelly; Steel, Gavin; Young, Jack; Paszkiewicz, Konrad; Millar, Andrew J.; Halliday, Karen J.; Hall, Anthony J.; Penfield, Steven

    2013-01-01

    Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock. PMID:24254125

  4. Isolation of three testis-specific genes (TSA303, TSA806, TSA903) by a differential mRNA display method

    SciTech Connect

    Ozaki, Kouichi; Kuroki, Tamotsu; Hayashi, Seitaku; Nakamura, Yusuke

    1996-09-01

    We isolated three human testis-specific genes by a differential mRNA display method. The cDNAs contained open reading frames of 1620, 453, and 333 nucleotides, encoding 540, 151, 111 amino acids, respectively. The first of these genes, designated TSA303, encodes a novel protein homologous to TCP20, one of the subunits of the human TRiC chaperonin complex that can bind newly synthesized or unstable folding intermediates of polypeptides and assist substrate proteins in folding, assembly, and transport. The second, TSA806, encodes a novel protein containing 3.3 contiguous repeats of the cdc10/swi6 (ankyrin) motif that was originally found in products of cell cycle control genes of yeast and cell fate determination genes in Drosophila and Caenorhabditis elegans. The third gene, TSA903, encodes a protein homologous to the C-terminal region of murine uridine monophosphate kinase. Northern blot analysis confirmed that in 16 human adult tissues examined, each of these genes was expressed specifically in the testis. From the results of cDNA screening of nearly 1 million plaques, the abundance of each transcript in a preparation of total mRNA was estimated as 0.0004% (TSA303), 0.0006% (TSA806), and 0.0002% (TSA903). Our results imply that the differential display method is a powerful tool for isolation of tissue-specific genes even if they are expressed at a level as low as 1 in several hundred thousand to a million molecules of total mRNA. 38 refs., 1 fig., 3 tabs.

  5. Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA

    PubMed Central

    Stamatopoulos, Basile; Haibe-Kains, Benjamin; Equeter, Carole; Meuleman, Nathalie; Sorée, Anne; De Bruyn, Cécile; Hanosset, Delphine; Bron, Dominique; Martiat, Philippe; Lagneaux, Laurence

    2009-01-01

    Background Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. Design and Methods In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Results Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70+ patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70+ patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70+ cells had better adhesion/migration capacities and only ZAP70+ patient cells responded to microenvironment contact by CXCR4 downregulation. Conclusions We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70+ cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease. PMID:19377082

  6. alpha-Amanitin-insensitive transcription of mouse beta major-globin 5'-flanking and structural gene sequences correlates with mRNA expression.

    PubMed Central

    Carlson, D P; Ross, J

    1984-01-01

    A small proportion of the RNAs of mouse reticulocytes consists of beta major-globin mRNA sequences linked to sequences transcribed from the 5'-flanking region of the beta major-globin gene. These upstream RNAs are polyadenylylated and contain 700-800 nucleotides, and their 5' regions are heterogeneous. RNAs with similar or identical 5' regions are transcribed in cell-free extracts from a circular mouse beta major-globin gene template. Synthesis of most of the upstream RNAs in vitro is not inhibited by low levels (1 microgram/ml) of alpha-amanitin, indicating that they are transcribed by an enzyme(s) different from RNA polymerase II. During culture of mouse erythroleukemia cells with dimethyl sulfoxide, globin mRNA and upstream RNAs accumulate with similar kinetics. In contrast, upstream RNAs are not detected in hemin-treated cells. Images PMID:6595660

  7. Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis.

    PubMed

    Pichard, Scott L; Paul, John H

    1991-06-01

    A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences. PMID:16348507

  8. Identification of Cyclobutane Pyrimidine Dimer-Responsive Genes Using UVB-Irradiated Human Keratinocytes Transfected with In Vitro-Synthesized Photolyase mRNA

    PubMed Central

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; van der Horst, Gijsbertus T. J.; Szegedi, Andrea; Horkay, Irén; Emri, Gabriella; Karikó, Katalin; Remenyik, Éva

    2015-01-01

    Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases. PMID:26121660

  9. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

    PubMed

    Márkus, Nóra M; Hasel, Philip; Qiu, Jing; Bell, Karen F S; Heron, Samuel; Kind, Peter C; Dando, Owen; Simpson, T Ian; Hardingham, Giles E

    2016-01-01

    Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms. PMID:26828201

  10. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling

    PubMed Central

    Márkus, Nóra M.; Hasel, Philip; Qiu, Jing; Bell, Karen F. S.; Heron, Samuel; Kind, Peter C.; Dando, Owen; Simpson, T. Ian; Hardingham, Giles E.

    2016-01-01

    Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms. PMID:26828201

  11. Nonsense mutations in the rhodopsin gene that give rise to mild phenotypes trigger mRNA degradation in human cells by nonsense-mediated decay.

    PubMed

    Roman-Sanchez, Ramon; Wensel, Theodore G; Wilson, John H

    2016-04-01

    Eight different nonsense mutations in the human rhodopsin gene cause retinitis pigmentosa (RP), an inherited degenerative disease of the retina that can lead to complete blindness. Although all these nonsense mutations lead to premature termination codons (PTCs) in rhodopsin mRNA, some display dominant inheritance, while others are recessive. Because nonsense-mediated decay (NMD) can degrade mRNAs containing PTCs and modulate the inheritance patterns of genetic diseases, we asked whether any of the nonsense mutations in the rhodopsin gene generated mRNAs that were susceptible to degradation by NMD. We hypothesized that nonsense mutations that caused mild RP phenotypes would trigger NMD, whereas those that did not engage NMD would cause more severe RP phenotypes-presumably due to the toxicity of the truncated protein. To test our hypothesis, we transfected human rhodopsin nonsense mutants into HEK293 and HT1080 human cells and measured transcript levels by qRT-PCR. In both cell lines, rhodopsin mutations Q64X and Q344X, which cause severe phenotypes that are dominantly inherited, yielded the same levels of rhodopsin mRNA as wild type. By contrast, rhodopsin mutations W161X and E249X, which cause recessive RP, showed decreased rhodopsin mRNA levels, consistent with NMD. Rhodopsin mutant Y136X, a dominant mutation that causes late-onset RP with a very mild pathology, also gave lower mRNA levels. Treatment of cells with Wortmannin, an inhibitor of NMD, eliminated the degradation of Y136X, W161X, and E249X rhodopsin mRNAs. These results suggest that NMD modulates the severity of RP in patients with nonsense mutations in the rhodopsin gene. PMID:26416182

  12. Chronic toxicity of pesticides to the mRNA expression levels of metallothioneins and cytochrome P450 1A genes in rainbow trout.

    PubMed

    Ceyhun, Saltuk Bugrahan; Aksakal, Ercüment; Kirim, Birsen; Atabeyoglu, Kübra; Erdogan, Orhan

    2012-03-01

    The hazardous effects of pesticides on various metabolic pathways are a great problem for environmental health and should be well determined. In the present study, the authors treated rainbow trout with 0.6 μg/L deltamethrin for 28 days and 1.6 mg/L 2,2-dichlorovinyl dimethyl phosphate for 21 days. After this time period, the authors observed alterations in mRNA expression levels of MT-A, MT-B and CYP-1A. Chronic exposure to low levels of pesticides may have a more significant effect on fish populations than acute poisoning. While both pesticides caused a significant increase on mRNA levels of MT-A and CYP-1A, MT-B mRNA levels were increased significantly only upon deltamethin administration. The significant increase in mRNA levels of the corresponding genes may be considered as a defence mechanism in addition to the antioxidants against oxidative stress, as well as a detoxification mechanism against adverse effects of pesticides. PMID:21665904

  13. The NR3C1 Glucocorticoid Receptor Gene Polymorphisms May Modulate the TGF-beta mRNA Expression in Asthma Patients.

    PubMed

    Panek, Michał; Pietras, Tadeusz; Fabijan, Artur; Zioło, Jan; Wieteska, Łukasz; Małachowska, Beata; Fendler, Wojciech; Szemraj, Janusz; Kuna, Piotr

    2015-08-01

    Glucocorticosteroids (GCs) are basic drugs in therapy of a number of diseases, including chronic diseases of the respiratory system. They are the most important anti-inflammatory drugs in the treatment of asthma. GCs after binding to the glucocorticoid receptor (GR) form the complex (transcription factor), which acts on promoter and regulatory parts of genes enhancing the expression of anti-inflammatory proteins and decreasing the proinflammatory protein synthesis, including numerous cytokines mediating inflammation in the course of asthma. Non-sensitivity or resistance to GCs favours an increase in the TGF-β expression. This cytokine plays a central role in asthma inducing fibroblast differentiation and extracellular matrix synthesis. TGF-β isoforms, 1, 2 and 3, are located on chromosome 19q13, 1q41 and 14q24, respectively. GCs reduce TGF-β 1 and TGF-β 2 production and significantly decrease the expression of upregulated TGF-β 1 and TGF-β 2 mRNA induced by exogenous TGF-β. In asthma, TGF-β may play a role in the development of the peribronchiolar and subepithelial fibrosis, which contributes to a significant clinical exacerbation of asthma. Therefore, it is possible that NR3C1 glucocorticoid receptor gene polymorphisms could exert varied effects on the TGF-β mRNA expression and fibrotic process in lungs of asthmatic patients. The aim of the study was to evaluate the impact of polymorphic forms (Tth111I, BclI, ER22/23EK, N363S) of the NR3C1 gene on the level of the TGF-β 1 mRNA expression. A total of 173 patients with asthma and 163 healthy volunteers participated in the study. Genotyping of Tth111I, BclI, ER22/23EK, and N363S polymorphisms of the NR3C1 gene was performed by using PCR-HRM and PCR-RFLP techniques. TGF-β mRNA was assessed by real time RT-PCR. Tth111I SNP significantly (p = 0.0115) correlated with the TGF-β 1 mRNA expression level. The significance of AA and GG genotypes of Tth111I SNP in increasing and decreasing the level of the TGF-β 1

  14. In vivo mRNA profiling of uropathogenic Escherichia coli from diverse phylogroups reveals common and group-specific gene expression profiles.

    PubMed

    Bielecki, Piotr; Muthukumarasamy, Uthayakumar; Eckweiler, Denitsa; Bielecka, Agata; Pohl, Sarah; Schanz, Ansgar; Niemeyer, Ute; Oumeraci, Tonio; von Neuhoff, Nils; Ghigo, Jean-Marc; Häussler, Susanne

    2014-01-01

    mRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression of Escherichia coli pathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associated E. coli isolates to different phylogenetic groups. Whereas the in vivo gene expression profiles of the majority of genes were conserved among 21 E. coli strains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribed in vivo relative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease. Importance: Urinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenic Escherichia coli strains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenic E. coli gene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host. PMID:25096872

  15. mRNA expression profile of multidrug-resistant genes in acute lymphoblastic leukemia of children, a prognostic value for ABCA3 and ABCA2.

    PubMed

    Rahgozar, Soheila; Moafi, Alireza; Abedi, Marjan; Entezar-E-Ghaem, Mansureh; Moshtaghian, Jamal; Ghaedi, Kamran; Esmaeili, Abolghasem; Montazeri, Fatemeh

    2014-01-01

    Multidrug resistance (MDR) is an important cause of treatment failure in acute lymphoblastic leukemia (ALL). The ABC family of membrane transporters is proposed, albeit with controversy, to be involved in this process. The present study aims to investigate the mRNA expression profile of several genes of this family, including ABCA2, ABCA3, ABCB1/MDR1, MRP1/ABCC1, MRP3/ABCC3, ABCG2/BCRP, and the intracellular transporter MVP/LRP, in childhood ALL, and to evaluate their association with response to therapy. Some genes in the present research are being studied for the first time in Iran. Using quantitative real-time PCR, we evaluated 27 children with ALL at diagnosis and 15 children with normal bone marrow. The status of response to therapy was assessed one year after the onset of therapy through investigating the IgH/TCRγ gene rearrangements. Our findings indicate a considerable and direct relationship between mRNA expression levels of ABCA2, ABCA3, MDR1, and MRP1 genes and positive minimal residual disease (MRD) measured after one year of treatment. Statistical analysis revealed that expression of these genes higher than the cutoff point will raise the risk of MRD by 15-, 6.25-, 12-, and 9-fold, respectively. No relationship was found between of MVP/LRP, MRP3 and ABCG2 genes expression and ALL prognoses. Considering the direct and significant relationship between the increased expression of ABCA2, ABCA3, MDR1, and MRP1 genes and positive risk of MRD in children with ALL, evaluating the expression profile of these genes on diagnosis may identify high risk individuals and help plan a more efficient treatment strategy. PMID:24145140

  16. Modulation of Ergot Alkaloids in a Grass-Endophyte Symbiosis by Alteration of mRNA Concentrations of an Ergot Alkaloid Synthesis Gene.

    PubMed

    Mulinti, Prashanthi; Florea, Simona; Schardl, Christopher L; Panaccione, Daniel G

    2016-06-22

    The profile of ergot alkaloids in perennial ryegrass (Lolium perenne) containing the endophytic fungus Epichloë typhina × festucae includes high concentrations of the early pathway metabolites ergotryptamine and chanoclavine-I in addition to the pathway end-product ergovaline. Because these alkaloids differ in activity, we investigated strategies to alter their relative concentrations. An RNAi-based approach reduced the concentration of mRNA from the gene easA, which encodes an enzyme required for a ring closure that separates ergotryptamine and chanoclavine-I from ergovaline. Lower easA mRNA concentrations correlated with lower concentrations of ergovaline and higher concentrations of ergotryptamine and chanoclavine-I. Overexpression of easA led to higher concentrations of ergovaline in leaf blades but not in pseudostems; concentrations of the early pathway metabolites were not altered in overexpression strains. The data indicate that altering the concentration of mRNA from a single gene can change alkaloid flux, but the magnitude of the change was limited and variable. PMID:27248330

  17. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain

    PubMed Central

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  18. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain.

    PubMed

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5'-upstream region. Using genotype imputation, "R(2)-Δ(2)" analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30-40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5'-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  19. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    PubMed

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  20. LDLR gene synonymous mutation c.1813C>T results in mRNA splicing variation in a kindred with familial hypercholesterolaemia.

    PubMed

    Ho, Clement K M; Musa, Fathel Rahman; Bell, Christine; Walker, Simon W

    2015-11-01

    Familial hypercholesterolaemia, one of the most common inherited diseases in the general population, is associated with mutations in at least three different genes including the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and protein convertase subtilisin/kexin type 9 (PCSK9) genes. In this report, we describe an unclassified DNA variant (c.1813C>T; p.Leu605Leu) within exon 12 of the LDLR gene in a kindred in which familial hypercholesterolaemia is associated with c.1813C>T heterozygosity. In silico analysis suggested that c.1813C>T might affect splicing of the LDLR gene by creating a cryptic donor splice site, which was confirmed by RT-PCR coupled with cDNA sequencing, to result in the loss of 34 base pairs in the coding sequence. The latter truncated mRNA is predicted to generate a frameshift leading to a premature stop at codon 652 and early termination of the low density lipoprotein receptor polypeptide chain, and thus provides a molecular basis for the hypercholesterolaemic phenotype. This case report highlights the emerging utility of RNA studies for the molecular diagnosis of familial hypercholesterolaemia in patients with potential mRNA splicing variants. PMID:25624525

  1. mRNA Levels of Imprinted Genes in Bovine In Vivo Oocytes, Embryos and Cross Species Comparisons with Humans, Mice and Pigs

    PubMed Central

    Jiang, Zongliang; Dong, Hong; Zheng, Xinbao; Marjani, Sadie L.; Donovan, David M.; Chen, Jingbo; Tian, Xiuchun (Cindy)

    2015-01-01

    Twenty-six imprinted genes were quantified in bovine in vivo produced oocytes and embryos using RNA-seq. Eighteen were detectable and their transcriptional patterns were: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); peaked at a specific stage (PHLDA2, SGCE, PEG10, PEG3, GNAS, MEG3, DGAT1, ASCL2, NNAT, and NAP1L5); or constantly low (DIRAS3, IGF2, H19 and RTL1). These patterns reflect mRNAs that are primarily degraded, important at a specific stage, or only required at low quantities. The mRNAs for several genes were surprisingly abundant. For instance, transcripts for the maternally imprinted MEST and PLAGL1, were high in oocytes and could only be expressed from the maternal allele suggesting that their genomic imprints were not yet established/recognized. Although the mRNAs detected here were likely biallelically transcribed before the establishment of imprinted expression, the levels of mRNA during these critical stages of development have important functional consequences. Lastly, we compared these genes to their counterparts in mice, humans and pigs. Apart from previously known differences in the imprinting status, the mRNA levels were different among these four species. The data presented here provide a solid reference for expression profiles of imprinted genes in embryos produced using assisted reproductive biotechnologies. PMID:26638780

  2. Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.

    PubMed

    Iizuka, Satoshi; Oridate, Nobuhiko; Nashimoto, Masayuki; Fukuda, Satoshi; Tamura, Masato

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs. PMID:25437003

  3. Alternative splicing of human T-cell-specific MAL mRNA and its correlation with the exon/intron organization of the gene

    SciTech Connect

    Rancano, C.; Rubio, T.; Alonso, M.A. )

    1994-05-15

    Sequence analysis of the T-cell-specific MAL gene revealed four exons, each encoding a hydrophobic, presumably membrane-associated, segment and its adjacent hydrophilic sequence. Amplification by the polymerase chain reaction of cDNA from different T-cell samples indicated the existence of four different forms of MAL mRNA, termed MAL-a, -b, -c, and -d, that arise from differential usage of exons II and/or III. As the three introns were located between complete codons, the reading frame was maintained in all the transcripts. A model resembling the structures postulated for different proteolipid proteins is proposed for the protein encoded by each alternative mRNA species. 9 refs., 3 figs.

  4. Bovine DNase I: gene organization, mRNA expression, and changes in the topological distribution of the protein during apoptosis in lens epithelial cells.

    PubMed

    De María, Alicia; Arruti, Cristina

    2003-12-19

    Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation. PMID:14680812

  5. RPL13A as a reference gene for normalizing mRNA transcription of ovarian cancer cells with paclitaxel and 10-hydroxycamptothecin treatments.

    PubMed

    Bian, Zehua; Yu, Yang; Quan, Chao; Guan, Rongwei; Jin, Yan; Wu, Jie; Xu, Lidan; Chen, Feng; Bai, Jing; Sun, Wenjing; Fu, Songbin

    2015-04-01

    Gene transcription analysis is important in cancer research, and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) has been demonstrated to be an effective method to evaluate gene transcription in cancer. RT‑qPCR requires an internal reference gene with a consistent level of mRNA transcription across various experimental conditions. However, it has been suggested that different treatments, including anticancer therapy, may influence the transcriptional stability of internal reference genes. Paclitaxel (PTX) and 10‑hydroxycamptothecin (HCPT) are widely used to treat various types of cancer, and a suitable internal reference gene is required in order to analyze the transcription profiles of the cells following treatment. In the current study, the transcriptional stability of 30 candidate reference genes was investigated in cancer cells following treatment with PTX and HCPT. The two ovarian cancer cell lines, UACC‑1598 and SKOV3, were treated with PTX and HCPT for 24 and 48 h, and the transcriptional levels of the candidate reference genes were subsequently evaluated by RT‑qPCR analysis. The transcriptional stability of the selected genes was then analyzed using qbase+ and NormFinder software. A total of 9 genes were demonstrated to exhibit high transcriptional stability and one of these genes, ribosomal protein L13a (RPL13A), was identified to exhibit high transcriptional stability in every group. The current study identified various reference genes suitable under different circumstances, while RPL13A was indicated to be the most suitable reference gene for analyzing the transcription profile of ovarian cancer cells following treatment with PTX and HCPT. PMID:25523336

  6. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken.

    PubMed

    Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

    2015-10-01

    This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

  7. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken

    PubMed Central

    Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

    2015-01-01

    This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

  8. Dietary sunflower oil modulates milk fatty acid composition without major changes in adipose and mammary tissue fatty acid profile or related gene mRNA abundance in sheep.

    PubMed

    Castro-Carrera, T; Frutos, P; Leroux, C; Chilliard, Y; Hervás, G; Belenguer, A; Bernard, L; Toral, P G

    2015-04-01

    There are very few studies in ruminants characterizing mammary and adipose tissue (AT) expression of genes and gene networks for diets causing variations in milk fatty acid (FA) composition without altering milk fat secretion, and even less complementing this information with data on tissue FA profiles. This work was conducted in sheep in order to investigate the response of the mammary gland and the subcutaneous and perirenal AT, in terms of FA profile and mRNA abundance of genes involved in lipid metabolism, to a diet known to modify milk FA composition. Ten lactating Assaf ewes were randomly assigned to two treatments consisting of a total mixed ration based on alfalfa hay and a concentrate (60 : 40) supplemented with 0 (control diet) or 25 (SO diet) g of sunflower oil/kg of diet dry matter for 7 weeks. Milk composition, including FA profile, was analysed after 48 days on treatments. On day 49, the animals were euthanized and tissue samples were collected to analyse FA and mRNA abundance of 16 candidate genes. Feeding SO did not affect animal performance but modified milk FA composition. Major changes included decreases in the concentration of FA derived from de novo synthesis (e.g. 12:0, 14:0 and 16:0) and increases in that of long-chain FA (e.g. 18:0, c9-18:1, trans-18:1 isomers and c9,t11-CLA); however, they were not accompanied by significant variations in the mRNA abundance of the studied lipogenic genes (i.e. ACACA, FASN, LPL, CD36, FABP3, SCD1 and SCD5) and transcription factors (SREBF1 and PPARG), or in the constituent FA of mammary tissue. Regarding the FA composition of AT, the little influence of SO did not appear to be linked to changes in gene mRNA abundance (decreases of GPAM and SREBF1 in both tissues, and of PPARG in the subcutaneous depot). Similarly, the great variation between AT (higher contents of saturated FA and trans-18:1 isomers in the perirenal, and of cis-18:1, c9,t11-CLA and n-3 PUFA in the subcutaneous AT) could not be related to

  9. TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine

    PubMed Central

    Yuan, Zhi-Yi; Lu, Xi; Lei, Fan; Chai, Yu-Shuang; Wang, Yu-Gang; Jiang, Jing-Fei; Feng, Tian-Shi; Wang, Xin-Pei; Yu, Xuan; Yan, Xiao-Jin; Xing, Dong-Ming; Du, Li-Jun

    2015-01-01

    Berberine (BBR) is a natural compound with variable pharmacological effects and a broad panel of target genes. We investigated berberine’s pharmacological activities from the perspective of its nucleotide-binding ability and discovered that BBR directly regulates gene expression by targeting TATA boxes in transcriptional regulatory regions as well as the poly adenine (poly (A)) tail at the mRNA terminus. BBR inhibits gene transcription by binding the TATA boxes in the transcriptional regulatory region, but it promotes higher levels of expression by targeting the poly (A) tails of mRNAs. The present study demonstrates that TATA boxes and poly (A) tails are the first and second primary targets by which BBR regulates gene expression. The final outcome of gene regulation by BBR depends on the structure of the individual gene. This is the first study to reveal that TATA boxes and poly (A) tails are direct targets for BBR in its regulation of gene expression. Our findings provide a novel explanation for the complex activities of a small molecule compound in a biological system and a novel horizon for small molecule-compound pharmacological studies. PMID:26671652

  10. Drug-induced mRNA signatures are enriched for the minority of genes that are highly heritable.

    PubMed

    Gao, Tianxiang; Brodin, Petter; Davis, Mark M; Jojic, Vladimir

    2015-01-01

    The blood gene expression signatures are used as biomarkers for immunological and non- immunological diseases. Therefore, it is important to understand the variation in blood gene expression patterns and the factors (heritable/non-heritable) that underlie this variation. In this paper, we study the relationship between drug effects on the one hand, and heritable and non-heritable factors influencing gene expression on the other. Understanding of this relationship can help select appropriate targets for drugs aimed at reverting disease phenotypes to healthy states. In order to estimate heritable and non-heritable effects on gene expression, we use Twin-ACE model on a gene expression dataset MuTHER, measured in blood samples from monozygotic and dizygotic twins. In order to associate gene expression with drug effects, we use CMap database. We show that, even though the expressions of most genes are driven by non-heritable factors, drugs are more likely to influence expression of genes, driven by heritable rather than non-heritable factors. We further study this finding in the context of a gene regulatory network. We investigate the relationship between the drug effects on gene expression and propagation of heritable and non-heritable factors through regulatory networks. We find that the decisive factor in determining whether a gene will be influenced by a drug is the flow of heritable effects supplied to the gene through regulatory network. PMID:25592599

  11. Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens.

    PubMed

    Maslov, D A; Hollar, L; Haghighat, P; Nawathean, P

    1998-06-01

    Maxicircle molecules of kDNA in several isolates of Phytomonas were detected by hybridization with the 12S rRNA gene probe from Leishmania tarentolae. The estimated size of maxicircles is isolate-specific and varies from 27 to 36 kb. Fully edited and polyadenylated mRNA for kinetoplast-encoded ribosomal protein S12 (RPS12) was found in the steady-state kinetoplast RNA isolated from Phytomonas serpens strain 1G. Two minicircles (1.45 kb) from this strain were also sequenced. Each minicircle contains two 120 bp conserved regions positioned 180 degrees apart, a region enriched with G and T bases and a variable region. One minicircle encodes a gRNA for the first block of editing of RPSl2 mRNA, and the other encodes a gRNA with unknown function. A gRNA gene for the second block of RPSl2 was found on a minicircle sequenced previously. On each minicircle, a gRNA gene is located in the variable region in a similar position and orientation with respect to the conserved regions. PMID:9662707

  12. The feathering gene is linked to degranulation and oxidative burst not cytokine/chemokine mRNA expression levels or Salmonella enteritidis organ invasion in broilers.

    PubMed

    Swaggerty, Christina L; He, Haiqi; Genovese, Kenneth J; Kaiser, Pete; Pevzner, Igal Y; Kogut, Michael H

    2006-12-01

    In the past, we showed differences in heterophil function between parental broilers (A [fast feathering] > B [slow feathering]) and their F1 reciprocal crosses (D [fast feathering] > C [slow feathering]). In the present study, we evaluated the linkage of the feathering gene to heterophil function, pro-inflammatory cytokine/chemokine mRNA expression levels, and resistance to Salmonella enteritidis organ invasion. Heterophils were isolated from 2-day-old chickens (C and D) separated into males and females - slow males and females (SM and SF), and fast males and females (FM and FF). Heterophil functions of degranulation and oxidative burst were measured. Heterophils from FF chickens (183+/-8.9) released more (P < 0.05) beta-d-glucuronidase (microM) than heterophils from SF chickens (149+/-3.7); FF heterophils (4.6 x 10(4)) generated a significantly (P < 0.05) greater oxidative burst (mean relative fluorescent units) compared with SF heterophils (4.2 x 10(4)). Interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were quantitated by real-time quantitative reverse transcriptase-polymerase chain reaction. No differences were observed between SM and FM or between SF and FF heterophils. Finally, 1-day-old chickens were administered S. enteritidis and liver/spleen organ invasion was quantitated. No differences were observed between the number of S. enteritidis-positive FF and SF chickens, but FM were significantly (P < 0.05) more resistant to S. enteritidis organ invasion than SM chickens. The data indicate degranulation and oxidative burst were linked with the feathering gene; however, interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were not. Furthermore, susceptibility to in vitro S. enteritidis organ invasion was not linked to the feathering gene. PMID:17121735

  13. Evaluation of mRNA Expression Levels of cyp51A and mdr1, Candidate Genes for Voriconazole Resistance in Aspergillus flavus

    PubMed Central

    Fattahi, Azam; Zaini, Farideh; Kordbacheh, Parivash; Rezaie, Sasan; Safara, Mahin; Fateh, Roohollah; Farahyar, Shirin; Kanani, Ali; Heidari, Mansour

    2015-01-01

    Background: Voriconazole Resistance (VRC-R) in Aspergillus flavus isolates impacts the management of aspergillosis, since azoles are the first choice for prophylaxis and therapy. However, to the best of our knowledge, the mechanisms underlying voriconazole resistance are poorly understood. Objectives: The present study was designed to evaluate mRNA expression levels of cyp51A and mdr1 genes in voriconazole resistant A. flavus by a Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique. Materials and Methods: Five A. flavus isolates with resistance to VRC were examined by a RT-PCR approach. Results: Four out of five isolates revealed cyp51A and mdr1 mRNA overexpression. Interestingly, the isolate, which was negative for cyp51A and mdr1 mRNA expression showed a high voriconazole Minimum Inhibitory Concentration (MIC). Furthermore, a computational-based analysis predicted that voriconazole resistance could be mediated through cooperation with a network protein interaction. Conclusions: Our experimental and in silico findings may provide new insight in the complex molecular pathways of drug resistance and also could assist design an efficient therapeutic strategy for aspergillosis treatment. PMID:26865941

  14. Identification by a Digital Gene Expression Displayer (DGED) and test by RT-PCR analysis of new mRNA candidate markers for colorectal cancer in peripheral blood.

    PubMed

    Lauriola, Mattia; Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone; Montroni, Isacco; Manaresi, Alessio; Zattoni, Davide; Rivetti, Stefano; Mattei, Gabriella; Coppola, Domenico; Strippoli, Pierluigi; Taffurelli, Mario; Solmi, Rossella

    2010-08-01

    Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the

  15. Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

    PubMed

    Benedito-Palos, Laura; Calduch-Giner, Josep A; Ballester-Lozano, Gabriel F; Pérez-Sánchez, Jaume

    2013-04-14

    The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA. PMID:22856503

  16. Transcriptional Autoregulation and Inhibition of mRNA Translation of Amino Acid Regulator Gene cpcA of Filamentous Fungus Aspergillus nidulans

    PubMed Central

    Hoffmann, Bernd; Valerius, Oliver; Andermann, Meike; Braus, Gerhard H.

    2001-01-01

    The CPCA protein of the filamentous fungus Aspergillus nidulans is a member of the c-Jun-like transcriptional activator family. It acts as central transcription factor of the cross-pathway regulatory network of amino acid biosynthesis and is functionally exchangeable for the general control transcriptional activator Gcn4p of Saccharomyces cerevisiae. In contrast to GCN4, expression of cpcA is strongly regulated by two equally important mechanisms with additive effects that lead to a fivefold increased CPCA protein amount under amino acid starvation conditions. One component of cpcA regulation involves a transcriptional autoregulatory mechanism via a CPCA recognition element (CPRE) in the cpcA promoter that causes a sevenfold increased cpcA mRNA level when cells are starved for amino acids. Point mutations in the CPRE cause a constitutively low mRNA level of cpcA and a halved protein level when amino acids are limited. Moreover, two upstream open reading frames (uORFs) in the 5′ region of the cpcA mRNA are important for a translational regulatory mechanism. Destruction of both short uORFs results in a sixfold increased CPCA protein level under nonstarvation conditions and a 10-fold increase under starvation conditions. Mutations in both the CPRE and uORF regulatory elements lead to an intermediate effect, with a low cpcA mRNA level but a threefold increased CPCA protein level independent of amino acid availability. These data argue for a combined regulation of cpcA that includes a translational regulation like that of yeast GCN4 as well as a transcriptional regulation like that of the mammalian jun and fos genes. PMID:11553722

  17. Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

    PubMed Central

    Wolff, Stephanie E.; Veldhoen, Nik; Helbing, Caren C.; Ramirez, Claire A.; Malpas, Janae M.; Propper, Catherine R.

    2015-01-01

    Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring for endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10−9 M) or 4-tert-octylphenol (OP; 10−9 M, 10−8 M, and 10−7 M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10−7 M OP increased testicular transcript levels. Treatment with 10−9 and 10−8 M OP, but not 10−7 M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed

  18. Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan.

    PubMed Central

    Grover, J; Roughley, P J

    1993-01-01

    The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8484718

  19. Zinc-bearing zeolite clinoptilolite improves tissue zinc accumulation in laying hens by enhancing zinc transporter gene mRNA abundance.

    PubMed

    Li, Linfeng; Li, Ping; Chen, Yueping; Wen, Chao; Zhuang, Su; Zhou, Yanmin

    2015-08-01

    A study was conducted to investigate effects of zinc-bearing zeolite clinoptilolite (ZnCP), as an alternative for zinc sulfate (ZnSO4), on laying performance, tissue Zn accumulation and Zn transporter genes expression in laying hens. Hy-Line Brown laying hens were allocated to three treatments, each of which had six replicates with 15 hens per replicate, receiving basal diet supplemented with ZnSO4 (control, 80 mg Zn/kg diet), 0.23% ZnCP (40.25 mg Zn/kg diet) and 0.46% ZnCP (80.50 mg Zn/kg diet) for 8 weeks, respectively. Compared with control, hens fed diet containing 0.23% ZnCP had similar Zn content in measured tissues (P > 0.05). A higher ZnCP inclusion (0.46%) enhanced Zn accumulation in liver (P < 0.05) and pancreas (P < 0.05). In addition, ZnCP inclusion increased blood iron (Fe) content (P < 0.05). ZnCP supplementation enhanced jejunal metallothionein-4 (MT-4) messenger RNA (mRNA) abundance (P < 0.05). ZnCP inclusion at a higher level (0.46%) increased mRNA expression of MT-4 in pancreas (P < 0.05) and zinc transporter-1 (ZnT-1) in jejunum (P < 0.05). The highest ZnT-2 mRNA abundance in jejunum was found in hens fed 0.23% ZnCP inclusion diet (P < 0.05). The results indicated that ZnCP reached a higher bioavailability as compared with ZnSO4 as evidenced by enhanced tissue Zn accumulation and Zn transporter genes expression. PMID:25597922

  20. Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

    PubMed

    Shigeto, Hajime; Nakatsuka, Keisuke; Ikeda, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Funabashi, Hisakage

    2016-08-16

    This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner. PMID:27458920

  1. Effects of Dietary Selenium Against Lead Toxicity on mRNA Levels of 25 Selenoprotein Genes in the Cartilage Tissue of Broiler Chicken.

    PubMed

    Gao, H; Liu, C P; Song, S Q; Fu, J

    2016-07-01

    The interactions between the essential element selenium (Se) and the toxic element lead (Pb) have been reported extensively; however, little is known about the effect of Se on Pb toxicity and the expression pattern of selenoproteins in the cartilage of chicken. To investigate the effects of Se on Pb toxicity and the messenger RNA (mRNA) expressions of selenoproteins in cartilage tissue, an in vitro study was performed on 1-day-old broiler chickens (randomly allocated into four groups) with diet of different concentration of Se and Pb. After 90 days, the meniscus cartilage and sword cartilage tissue were examined for the mRNA levels of 25 selenoprotein genes. The results showed that Se and Pb influenced the expression of selenoprotein genes in the chicken cartilage tissue. In detail, Se could alleviate the downtrend of the expression of Gpx1, Gpx2, Gpx4, Txnrd2, Txnrd3, Dio1, Dio2, Seli, Selu, Sepx1, Selk, Selw, Selo, Selm, Sep15, Sepnn1, Sels, and Selt induced by Pb exposure in the meniscus cartilage. In the sword cartilage, Se alleviated the downtrend of the expression of Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Dio2, Dio3, Seli, Selh, SPS2, Sepx1, Selk, Selw, Selo, Selm, Sep15, Selpb, Sepn1, and Selt induced by Pb exposure. The present study provided some compensated data about the roles of Se against Pb toxicity in the regulation of selenoprotein expression. PMID:26643179

  2. A Large Number of Nuclear Genes in the Human Parasite Blastocystis Require mRNA Polyadenylation to Create Functional Termination Codons

    PubMed Central

    Klimeš, Vladimír; Gentekaki, Eleni; Roger, Andrew J.; Eliáš, Marek

    2014-01-01

    Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3′-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3′-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left. PMID:25015079

  3. The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

    PubMed Central

    Gallie, D R; Young, T E

    1994-01-01

    Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates. PMID:7824660

  4. Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene, decreased leptin mRNA expression, and decreased leptin serum levels

    PubMed Central

    Kloiber, Stefan; Ripke, Stephan; Kohli, Martin A.; Reppermund, Simone; Salyakina, Daria; Uher, Rudolf; McGuffin, Peter; Perlis, Roy H.; Hamilton, Steven P.; Pütz, Benno; Hennings, Johannes; Brückl, Tanja; Klengel, Torsten; Bettecken, Thomas; Ising, Marcus; Uhr, Manfred; Dose, Tatjana; Unschuld, Paul G.; Zihl, Josef; Binder, Elisabeth; Müller-Myhsok, Bertram; Holsboer, Florian; Lucae, Susanne

    2013-01-01

    Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9 × 10−5) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant

  5. Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene, decreased leptin mRNA expression, and decreased leptin serum levels.

    PubMed

    Kloiber, Stefan; Ripke, Stephan; Kohli, Martin A; Reppermund, Simone; Salyakina, Daria; Uher, Rudolf; McGuffin, Peter; Perlis, Roy H; Hamilton, Steven P; Pütz, Benno; Hennings, Johannes; Brückl, Tanja; Klengel, Torsten; Bettecken, Thomas; Ising, Marcus; Uhr, Manfred; Dose, Tatjana; Unschuld, Paul G; Zihl, Josef; Binder, Elisabeth; Müller-Myhsok, Bertram; Holsboer, Florian; Lucae, Susanne

    2013-07-01

    Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9×10(-5)) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR(⁎)D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant

  6. Gene microarray analysis of lncRNA and mRNA expression profiles in patients with hypopharyngeal squamous cell carcinoma

    PubMed Central

    Zhou, Jieyu; Li, Wenming; Jin, Tong; Xiang, Xuan; Li, Maocai; Wang, Juan; Li, Guojun; Pan, Xinliang; Lei, Dapeng

    2015-01-01

    Background: Studies have shown that long noncoding RNAs (lncRNAs) are involved in the development and progression of many types of cancer. However, the mechanisms by which lncRNAs influence development and progression of hypopharyngeal squamous cell carcinoma (HSCC) are unclear. Method: We investigated differences in lncRNA and mRNA expression profiles between 3 pairs of HSCC tissues and adjacent nontumor tissues by microarray analysis. Results: In HSCC tissues, 1299 lncRNAs were significantly upregulated (n=669) or downregulated (n=630) compared to levels in adjacent nontumor tissues. Moreover, 1432 mRNAs were significantly upregulated (n=684) or downregulated (n=748) in HSCC tissues. We randomly selected 2 differentially expressed lncRNAs (AB209630, AB019562) and 2 differentially expressed mRNAs (SPP1, TJP2) for confirmation of microarray results using qRT-PCR. The qRT-PCR results matched well with the microarray data. The differentially expressed lncRNAs and mRNAs were distributed on each of the chromosomes, including the X and Y chromosomes. Pathway analysis indicated that the biological functions of differentially expressed mRNAs were related to 48 cellular pathways that may be associated with HSCC development. GO analysis revealed that 593 mRNAs involved in biological processes, 50 mRNAs involved in cellular components, and 46 mRNAs involved in molecular functions were upregulated in the carcinomas; 280 mRNAs involved in biological processes, 58 mRNAs involved in cellular components, and 71 mRNAs involved in molecular functions were downregulated in the carcinomas. In addition, 8 enhancer-like lncRNAs and 21 intergenic lncRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Conclusion: These findings provide insight into the mechanisms underlying HSCC tumorigenesis and will facilitate identification of new therapeutic targets and diagnostic biomarkers for this disease. PMID:26131061

  7. Quantitative mRNA expression analysis of selected genes in patients with early-stage hypothyroidism induced by treatment with iodine-131.

    PubMed

    Guo, Kun; Gao, Rui; Yu, Yan; Zhang, Weixiao; Yang, Yuxuan; Yang, Aimin

    2015-11-01

    The present study aimed to investigate the molecular markers indicative of early-stage hypothyroidism induced by treatment with iodine-131, in order to assist in further investigations of radio iodine‑induced hypothyroidism. A total of 59 patients diagnosed with hyperthyroidism (male/female, 16/43; median age, 46.4 years) and 27 healthy subjects (male/female, 7/21; median age, 44.6 years) were included in the present study. All patients were treated with appropriate doses of iodine‑131 and, three months following treatment, the patients were subdivided into two groups: A group with early‑stage hypothyroidism symptoms, and a group with non‑early‑stage hypothyroidism, including euthyroid patients and patients remaining with hyperthyroidism. Tissue samples from the patients and healthy subjects were collected by fine needle biopsies, and the mRNA expression levels of B-cell lymphoma 2 (Bcl‑2), nuclear factor (NF)‑κB, Ku70, epidermal growth factor receptor (EGFR), early growth response 1 (Egr‑1), TP53 and ataxia telangiectasia mutated were analyzed using reverse transcription‑quantitative polymerase chain reaction prior to iodine‑131 treatment. The association of the variation of target genes with susceptibility to early‑stage hypothyroidism was analyzed. Compared with normal subjects, the mRNA expression levels of Ku70 (0.768, vs. 3.304, respectively; P<0.001) and EGFR (0.859, vs. 1.752, respectively; P<0.05) were significantly higher, whereas those of NF‑κB (0.884, vs. 0.578, respectively; P<0.05) and Bcl‑2 (1.235, vs. 0.834, respectively; P<0.05) were lower in the hyperthyroid patients. Following treatment with iodine‑131, 30 of the 59 (50.8%) patients with hyperthyroidism were diagnosed with early‑stage hypothyroidism, and in the early‑stage hypothyroidism group, the mRNA expression levels of Bcl‑2 were significantly decreased (P<0.05), whereas those of Egr‑1 (P<0.05) were significantly increased, compared with the non

  8. Sequence polymorphism of GroEL gene in natural population of Bacillus and Brevibacillus spp. that showed variation in thermal tolerance capacity and mRNA expression.

    PubMed

    Sen, R; Tripathy, S; Padhi, S K; Mohanty, S; Maiti, N K

    2014-10-01

    GroEL, a class I chaperonin, plays an important role in the thermal adaptation of the cell and helps to maintain the viability of the cell under heat shock condition. Function of groEL in vivo depends on the maintenance of proper structure of the protein which in turn depends on the nucleotide and amino acid sequence of the gene. In this study, we investigated the changes in nucleotide and amino acid sequences of the partial groEL gene that may affect the thermotolerance capacity as well as mRNA expression of bacterial isolates. Sequences among the same species having differences in the amino acid level were identified as different alleles. The effect of allelic variation on the groEL gene expression was analyzed by comparison and relative quantification in each allele under thermal shock condition by RT-PCR. Evaluation of K a/K s ratio among the strains of same species showed that the groEL gene of all the species had undergone similar functional constrain during evolution. The strains showing similar thermotolerance capacity was found to carry same allele of groEL gene. The isolates carrying allele having amino acid substitution inside the highly ATP/ADP or Mg(2+)-binding region could not tolerate thermal stress and showed lower expression of the groEL gene. Our results indicate that during evolution of these bacterial species the groEL gene has undergone the process of natural selection, and the isolates have evolved with the groEL allelic sequences that help them to withstand the thermal stress during their interaction with the environment. PMID:24894903

  9. RNA-binding protein regulates plant DNA methylation by controlling mRNA processing at the intronic heterochromatin-containing gene IBM1

    PubMed Central

    Wang, Xingang; Duan, Cheng-Guo; Tang, Kai; Wang, Bangshing; Zhang, Huiming; Lei, Mingguang; Lu, Kun; Mangrauthia, Satendra K.; Wang, Pengcheng; Zhao, Yang; Zhu, Jian-Kang

    2013-01-01

    DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1. Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3′ distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements. PMID:24003136

  10. Dietary Fucoxanthin Increases Metabolic Rate and Upregulated mRNA Expressions of the PGC-1alpha Network, Mitochondrial Biogenesis and Fusion Genes in White Adipose Tissues of Mice

    PubMed Central

    Wu, Meng-Ting; Chou, Hong-Nong; Huang, Ching-jang

    2014-01-01

    The mechanism for how fucoxanthin (FX) suppressed adipose accumulation is unclear. We aim to investigate the effects of FX on metabolic rate and expressions of genes related to thermogenesis, mitochondria biogenesis and homeostasis. Using a 2 × 2 factorial design, four groups of mice were respectively fed a high sucrose (50% sucrose) or a high-fat diet (23% butter + 7% soybean oil) supplemented with or without 0.2% FX. FX significantly increased oxygen consumption and carbon dioxide production and reduced white adipose tissue (WAT) mass. The mRNA expressions of peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), cell death-inducing DFFA-like effecter a (CIDEA), PPARα, PPARγ, estrogen-related receptor α (ERRα), β3-adrenergic receptor (β3-AR) and deiodinase 2 (Dio2) were significantly upregulated in inguinal WAT (iWAT) and epididymal WAT (eWAT) by FX. Mitochondrial biogenic genes, nuclear respiratory factor 1 (NRF1) and NRF2, were increased in eWAT by FX. Noticeably, FX upregulated genes of mitochondrial fusion, mitofusin 1 (Mfn1), Mfn2 and optic atrophy 1 (OPA1), but not mitochondrial fission, Fission 1, in both iWAT and eWAT. In conclusion, dietary FX enhanced the metabolic rate and lowered adipose mass irrespective of the diet. These were associated with upregulated genes of the PGC-1α network and mitochondrial fusion in eWAT and iWAT. PMID:24534841

  11. Association between mRNA expression of chemotherapy-related genes and clinicopathological features in colorectal cancer: A large-scale population analysis.

    PubMed

    Shimamoto, Yuji; Nukatsuka, Mamoru; Takechi, Teiji; Fukushima, Masakazu

    2016-02-01

    To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right‑ and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right‑sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was

  12. Association between mRNA expression of chemotherapy-related genes and clinicopathological features in colorectal cancer: A large-scale population analysis

    PubMed Central

    SHIMAMOTO, YUJI; NUKATSUKA, MAMORU; TAKECHI, TEIJI; FUKUSHIMA, MASAKAZU

    2016-01-01

    To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right- and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right-sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was

  13. Carnitine palmitoyltransferase I gene in Synechogobius hasta: Cloning, mRNA expression and transcriptional regulation by insulin in vitro.

    PubMed

    Wu, Kun; Zheng, Jia-Lang; Luo, Zhi; Chen, Qi-Liang; Zhu, Qing-Ling; Wei-Hu

    2016-01-15

    We cloned seven complete CPT I cDNA sequences (CPT I α1a-1a, CPT I α1a-1b, CPT I α1a-1c, CPT I α1a-2, CPT I α2a, CPT I α2b1a, CPT I β) and a partial cDNA sequence (CPT I α2b1b) from Synechogobius hasta. Phylogenetic analysis shows that there are four CPT I duplications in S. hasta, CPT I duplication resulting in CPT I α and CPT I β, CPT I α duplication producing CPT I α1 and CPT I α2, CPT I α2 duplication generating CPT I α2a and CPT I α2b, and CPT I α2b duplication creating CPT I α2b1a and CPT I α2b1b. Alternative splicing of CPT Iα1a results in the generation of four CPT I isoforms, CPT I α1a-1a, CPT I α1a-1b, CPT I α1a-1c and CPT I α1a-2. Five CPT I transcripts (CPT I α1a, CPT I α2a, CPT I α2b1a, CPT I α2b1b and CPT I β) mRNAs are expressed in a wide range of tissues, but their abundance of each CPT I mRNA shows the tissue-dependent expression patterns. Insulin incubation significantly reduces the mRNA expression of CPT Iα1a and CPT Iα2a, but not other transcripts in hepatocytes of S. hasta. For the first time, our study demonstrates CPT Iα2b duplication and CPT I α1a alternative splicing in fish at transcriptional level, and the CPT I mRNAs are differentially regulated by insulin in vitro, suggesting that four CPT I isoforms may play different physiological roles during insulin signaling. PMID:26506441

  14. Methylation of the tryptophan hydroxylase‑2 gene is associated with mRNA expression in patients with major depression with suicide attempts.

    PubMed

    Zhang, Yuqi; Chang, Zaohuo; Chen, Jionghua; Ling, Yang; Liu, Xiaowei; Feng, Zhang; Chen, Caixia; Xia, Minghua; Zhao, Xingfu; Ying, Wang; Qing, Xu; Li, Guilin; Zhang, Changsong

    2015-08-01

    Tryptophan hydroxylase-2 (TPH2) contributes to alterations in the function of neuronal serotonin (5-HT), which are associated with various psychopathologies, including major depressive disorder (MDD) or suicidal behavior. The methylation of a single CpG site in the promoter region of TPH2 affects gene expression. Suicide and MDD are strongly associated and genetic factors are at least partially responsible for the variability in suicide risk. The aim of the present study was to investigate whether variations in TPH2 methylation in peripheral blood samples may predispose patients with MDD to suicide attempts. TPH2 mRNA expression levels differed significantly between 50 patients with MDD who had attempted suicide (MDD + suicide group) and 75 control patients with MDD (MDD group); TPH2 expression levels were significantly decreased (P=0.0005) in the patients who had attempted suicide. Furthermore, the frequency of TPH2 methylation was 36.0% in the MDD + suicide group, while it was 13.0% in the MDD group. The results of the present study demonstrated that methylation in the promoter region of TPH2 significantly affected the mRNA expression levels of TPH2, thus suggesting that methylation of the TPH2 promoter may silence TPH2 mRNA expression in MDD patients with or without suicidal behavior. In addition, there was a significant correlation between the methylation status of the TPH2 promoter and depression, hopelessness and cognitive impairment in the MDD + suicide group. In conclusion, the present study demonstrated that TPH2 expression was regulated by DNA methylation of the TPH2 promoter region in patients with MDD. PMID:25955598

  15. Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro.

    PubMed

    Rossi, Rodrigo O D S; da Cunha, Ellen V; Portela, Antonia M L R; Passos, José R S; Costa, José J N; Silva, Anderson W B; Saraiva, Márcia V A; Peixoto, Christina A; Donato, Mariana A M; van den Hurk, Robert; Silva, José R V

    2016-03-01

    This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (P<0.05) increase in follicular diameter after 18 days of culture. However, both FSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5. PMID:26435174

  16. The arabidopsis polyamine transporter LHRI/AtPUT3 modulates heat responsive gene expression by regulating mRNA stability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyamines (PA) involve in the gene regulation by interacting with various anionic macromolecules such as DNA, RNA and proteins and modulating their structure and function. Previous studies have showed that changing in polyamine biosynthesis alters plant response to different abiotic stresses. Here,...

  17. Calmodulin Gene Family in Potato: Developmental and Touch-Induced Expression of the mRNA Encoding a Novel Isoform

    NASA Technical Reports Server (NTRS)

    Takezawa, D.; Liu, Z. H.; An, G.; Poovaiah, B. W.

    1995-01-01

    Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

  18. Lesions in the mRNA cap-binding gene ABA HYPERSENSITIVE 1 suppress FRIGIDA-mediated delayed flowering in Arabidopsis.

    PubMed

    Bezerra, Isabel C; Michaels, Scott D; Schomburg, Fritz M; Amasino, Richard M

    2004-10-01

    Recessive mutations that suppress the late-flowering phenotype conferred by FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) and which also result in serrated leaf morphology were identified in T-DNA and fast-neutron mutant populations. Molecular analysis showed that the mutations are caused by lesions in the gene encoding the large subunit of the nuclear mRNA cap-binding protein, ABH1 (ABA hypersensitive1). The suppression of late flowering is caused by the inability of FRI to increase FLC mRNA levels in the abh1 mutant background. The serrated leaf morphology of abh1 is similar to the serrate (se) mutant and, like abh1, se is also a suppressor of FRI-mediated late flowering although it is a weaker suppressor than abh1. Unlike se, in abh1 the rate of leaf production and the number of juvenile leaves are not altered. The abh1 lesion affects several developmental processes, perhaps because the processing of certain mRNAs in these pathways is more sensitive to loss of cap-binding activity than the majority of cellular mRNAs. PMID:15361145

  19. Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress, hepatic stellate cell activation, cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs.

    PubMed

    Abhilash, P A; Harikrishnan, R; Indira, M

    2012-02-01

    Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β(1), TNF-α and α(1)(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β(1), TNF-α and α(1) (I) collagen in hepatic tissues. PMID:22149461

  20. The effect of lysophosphatidic acid during in vitro maturation of bovine oocytes: embryonic development and mRNA abundances of genes involved in apoptosis and oocyte competence.

    PubMed

    Boruszewska, Dorota; Torres, Ana Catarina; Kowalczyk-Zieba, Ilona; Diniz, Patricia; Batista, Mariana; Lopes-da-Costa, Luis; Woclawek-Potocka, Izabela

    2014-01-01

    In the present study we examined whether LPA can be synthesized and act during in vitro maturation of bovine cumulus oocyte complexes (COCs). We found transcription of genes coding for enzymes of LPA synthesis pathway (ATX and PLA2) and of LPA receptors (LPAR 1-4) in bovine oocytes and cumulus cells, following in vitro maturation. COCs were matured in vitro in presence or absence of LPA (10(-5) M) for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance of FST and GDF9 in oocytes and decreased mRNA abundance of CTSs in cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels of BCL2 and lower transcription levels of BAX resulting in the significantly lower BAX/BCL2 ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhanced in vitro development until the blastocyst stage, improved oocyte competence may be relevant for subsequent in vivo survival. PMID:24729661

  1. Comparison of the Expression Profiles of Immune Response Gene mRNA in Umbilical and Venous Blood of Newborns of the First Day of Life.

    PubMed

    Nepsha, O S; Nikitina, I V; Donnikov, A E; Bystritsky, A A; Mullabaeva, S M; Pavlovich, S V; Aleksandrova, N V

    2016-04-01

    The expression of immune response gene mRNA in the umbilical and venous blood were compared in newborns of the first day of life with and without signs of infection. The expression of il1b, il6, il8, il10, il12a, il15, il18, tnfa, tgfb1, tbx21, gata3, foxp3, rorc2, cd45, cd68, cd69, tlr2, tlr4, tlr9, and mmp8 mRNA was evaluated in umbilical and venous blood cells of newborns by reverse transcription real time PCR. In full-term newborns without signs of infection, the expression of il8, tlr2, tlr4, and mmp8 in venous blood was higher than in umbilical blood, while in preterm newborns, the levels of mmp8 transcript were elevated while the levels of tlr9, cd45, and gata3 were reduced. The expression of some markers differed in the umbilical and venous blood and in newborns with congenital infectious disease and without signs of infection. PMID:27165068

  2. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis

    PubMed Central

    Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2015-01-01

    Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination. PMID:26135744

  3. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

    PubMed

    Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2015-01-01

    Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination. PMID:26135744

  4. ADAR2 affects mRNA coding sequence edits with only modest effects on gene expression or splicing in vivo.

    PubMed

    Dillman, Allissa A; Cookson, Mark R; Galter, Dagmar

    2016-01-01

    Adenosine deaminases bind double stranded RNA and convert adenosine to inosine. Editing creates multiple isoforms of neurotransmitter receptors, such as with Gria2. Adar2 KO mice die of seizures shortly after birth, but if the Gria2 Q/R editing site is mutated to mimic the edited version then the animals are viable. We performed RNA-Seq on frontal cortices of Adar2(-/-) Gria2(R/R) mice and littermates. We found 56 editing sites with significantly diminished editing levels in Adar2 deficient animals with the majority in coding regions. Only two genes and 3 exons showed statistically significant differences in expression levels. This work illustrates that ADAR2 is important in site-specific changes of protein coding sequences but has relatively modest effects on gene expression and splicing in the adult mouse frontal cortex. PMID:26669816

  5. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm‑2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  6. Coordination of m(6)A mRNA Methylation and Gene Transcription by ZFP217 Regulates Pluripotency and Reprogramming.

    PubMed

    Aguilo, Francesca; Zhang, Fan; Sancho, Ana; Fidalgo, Miguel; Di Cecilia, Serena; Vashisht, Ajay; Lee, Dung-Fang; Chen, Chih-Hung; Rengasamy, Madhumitha; Andino, Blanca; Jahouh, Farid; Roman, Angel; Krig, Sheryl R; Wang, Rong; Zhang, Weijia; Wohlschlegel, James A; Wang, Jianlong; Walsh, Martin J

    2015-12-01

    Epigenetic and epitranscriptomic networks have important functions in maintaining the pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However, the mechanisms integrating the actions of these distinct networks are only partially understood. Here we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates the transcription of key pluripotency genes, and modulates N6-methyladenosine (m(6)A) deposition on their transcripts by sequestering the enzyme m(6)A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m(6)A RNA levels, and enhances m(6)A modification of the Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3 associated, and is enriched at promoters of m(6)A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m(6)A RNA modification to ensure ESC identity. PMID:26526723

  7. Effect of insoluble fibre on intestinal morphology and mRNA expression pattern of inflammatory, cell cycle and growth marker genes in a piglet model.

    PubMed

    Schedle, Karl; Pfaffl, Michael W; Plitzner, Christian; Meyer, Heinrich H D; Windisch, Wilhelm

    2008-12-01

    The effects of insoluble dietary fibre differing in lignin content on intestinal morphology and mRNA expression was tested in an animal model of 48 weaned piglets. Engaged fibre sources were wheat bran (rich in cellulose and hemicellulose) and pollen from Chinese Masson pine (Pinus massoniana) (rich in lignin), respectively. The fibre sources were added to a basal diet as follows: no addition (control), 3.0% wheat bran, 1.27% pine pollen, and 2.55% pine pollen. The 12 animals of each feeding group were fed four experimental diets ad libitum for 37 days and were then slaughtered for retrieving tissue samples from stomach, jejunum, ileum, colon and mesenterial lymph nodes. Both fibre sources increased villus height of mucosa in jejunum (+10% on average) and ileum (+16% on average). Results of mRNA expression rates of inflammatory, cell cycle and growth marker genes (NFkappaB, TNFalpha, TGFbeta, Caspase3, CDK4, IGF1) were specific to fibre source and tissue: wheat bran induced an up-regulation of NFkappaB in stomach and jejunum, as well as TNFalpha and TGFbeta, and Caspase3 in jejunum. Pine pollen induced down regulation of NFkappaB, TNFalpha, TGFbeta, Caspase3, CDK4 and IGF1 in the colon as well as up-regulation of NFkappaB and TGFbeta in mesenterial lymph nodes. Finally, an overall data comparison based on a hierarchical cluster analysis showed a close relation between gene regulation in different gut sections and organs, as well as between small intestine morphology and zootechnical performance. PMID:19143227

  8. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    PubMed

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  9. Two single nucleotide polymorphisms in the human nescient helix-loop-helix 2 (NHLH2) gene reduce mRNA stability and DNA binding.

    PubMed

    Al Rayyan, Numan; Wankhade, Umesh D; Bush, Korie; Good, Deborah J

    2013-01-01

    Nescient helix-loop-helix-2 (NHLH2) is a basic helix-loop-helix transcription factor, which has been implicated, using mouse knockouts, in adult body weight regulation and fertility. A scan of the known single nucleotide polymorphisms (SNPs) in the NHLH2 gene revealed one in the 3' untranslated region (3'UTR), which lies within an AUUUA RNA stability motif. A second SNP is nonsynonymous within the coding region of NHLH2, and was found in a genome-wide association study for obesity. Both of these SNPs were examined for their effect on NLHL2 by creating mouse mimics and examining mRNA stability, and protein function in mouse hypothalamic cell lines. The 3'UTR SNP causes increased instability and, when the SNP-containing Nhlh2 3'UTR is attached to luciferase mRNA, reduced protein levels in cells. The nonsynonymous SNP at position 83 in the protein changes an alanine residue, conserved in NHLH2 orthologs through the Drosophila sp. to a proline residue. This change affects migration of the protein on an SDS-PAGE gel, and appears to alter secondary structure of the protein, as predicted using in silico methods. These results provide functional information on two rare human SNPs in the NHLH2 gene. One of these has been linked to human obese phenotypes, while the other is present in a relatively high proportion of individuals. Given their effects on NHLH2 protein levels, both SNPs deserve further analysis in whether they are causative and/or additive for human body weight and fertility phenotypes. PMID:23026212

  10. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  11. Influence of functional polymorphisms in TNF-α, IL-8, and IL-10 cytokine genes on mRNA expression levels and risk of gastric cancer.

    PubMed

    de Oliveira, Juliana Garcia; Rossi, Ana Flávia Teixeira; Nizato, Daniela Manchini; Cadamuro, Aline Cristina Targa; Jorge, Yvana Cristina; Valsechi, Marina Curado; Venâncio, Larissa Paola Rodrigues; Rahal, Paula; Pavarino, Érika Cristina; Goloni-Bertollo, Eny Maria; Silva, Ana Elizabete

    2015-12-01

    Functional polymorphisms in promoter regions can produce changes in the affinity of transcription factors, thus altering the messenger ribonucleic acid (mRNA) expression levels of inflammatory cytokines associated with the risk of cancer development. The goal of this study was to evaluate the influence that polymorphisms in the cytokine genes known as TNF-α-308 G/A (rs1800629), TNF-α-857 C/T (rs1799724), IL-8-251 T/A (rs4073), IL-8-845 T/C (rs2227532), and IL-10-592 C/A (rs1800872) have on changes to mRNA expression levels and on the risks of chronic gastritis (CG) and gastric cancer (GC). A sample of 723 individuals was genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Relative mRNA expression levels were measured using quantitative real-time PCR (qPCR). Polymorphisms TNF-α-308 G/A and IL-8-251 A/T were not associated with risks of these gastric lesions. However, TNF-α-857 C/T, IL-8-845 T/C, and IL-10-592 C/A were found to be associated with a higher risk of GC, and IL-10-592 C/A was found to be associated with a higher risk of CG. The relative mRNA expression levels (RQ) of TNF-α, IL-8, and IL-10 were markedly downregulated in the CG group (median RQs = 0.128, 0.247, and 0.614, respectively), while the RQ levels of TNF-α in the GC group were upregulated (RQ = 2.749), but were basal for IL-8 (RQ = 1.053) and downregulated for IL-10 (RQ = 0.179). When the groups were stratified according to wild-type and polymorphic alleles, only for IL-8-845 T/C the polymorphic allele was found to influence the expression levels of this cytokine. IL-8-845 C allele carriers were significantly upregulated in both groups (GC and CG; RQ = 3.138 and 2.181, respectively) when compared to TT homozygotes (RQ = -0.407 and 0.165, respectively). In silico analysis in the IL-8 promoter region revealed that the presence of the variant C allele in position -845 is responsible for the presence of the binding

  12. The imprinted SNRPN gene is associated with a polycistronic mRNA and an imprinting control element

    SciTech Connect

    Saitoh, S.; Nicholls, R.D.; Seip, J.

    1994-09-01

    The small nuclear ribonucleoprotein-associated protein SmN (SNRPN) gene is located in the Prader-Willi syndrome (PWS) critical region in chromosome 15q11-q13. We have previously shown that it is functionally imprinted in humans, being only expressed from the paternal allele and differentially methylated on parental alleles. Therefore, SNRPN may have a role in PWS, although genetic studies suggest that at least two genes may be necessary for the classical PWS phenotype. We have characterized the SNRPN genomic structure, and shown that it comprises ten exons. Surprisingly, we identified an open reading frame (ORF) in the first three exons, 190-bp 5{prime} to the SmN ORF. Notably, the majority of base substitutions bewteen human and rodents in the upstream ORF occurred in the wobble position of codons, suggesting selection for a protein coding function. This ORF, which we name SNURF (SNRPN upstream reading frame) encodes a putative polypeptide of 71 amino acids. By analogy to prokaryotic operons that encode proteins with related functions, it is possible that SNURF may have a role in pre-mRNA splicing.

  13. Deep mRNA Sequencing of the Tritonia diomedea Brain Transcriptome Provides Access to Gene Homologues for Neuronal Excitability, Synaptic Transmission and Peptidergic Signalling

    PubMed Central

    Senatore, Adriano; Edirisinghe, Neranjan; Katz, Paul S.

    2015-01-01

    Background The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia), has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level. Results We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes). BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis) revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA) produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA. Conclusions Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain. PMID:25719197

  14. To translate, or not to translate: viral and host mRNA regulation by interferon-stimulated genes.

    PubMed

    Li, Melody M H; MacDonald, Margaret R; Rice, Charles M

    2015-06-01

    Type I interferon (IFN) is one of the first lines of cellular defense against viral pathogens. As a result of IFN signaling, a wide array of IFN-stimulated gene (ISG) products is upregulated to target different stages of the viral life cycle. We review recent findings implicating a subset of ISGs in translational regulation of viral and host mRNAs. Translation inhibition is mediated either by binding to viral RNA or by disrupting physiological interactions or levels of the translation complex components. In addition, many of these ISGs localize to translationally silent cytoplasmic granules, such as stress granules and processing bodies, and intersect with the microRNA (miRNA)-mediated silencing pathway to regulate translation of cellular mRNAs. PMID:25748385

  15. Effects of feed contaminant deoxynivalenol on plasma cytokines and mRNA expression of immune genes in the intestine of broiler chickens.

    PubMed

    Ghareeb, Khaled; Awad, Wageha A; Soodoi, Chimidtseren; Sasgary, Soleman; Strasser, Alois; Böhm, Josef

    2013-01-01

    An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1β, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κβ1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1β, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1β, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κβ1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes. PMID:23977054

  16. Effects of Feed Contaminant Deoxynivalenol on Plasma Cytokines and mRNA Expression of Immune Genes in the Intestine of Broiler Chickens

    PubMed Central

    Soodoi, Chimidtseren; Sasgary, Soleman; Strasser, Alois; Böhm, Josef

    2013-01-01

    An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1β, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κβ1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1β, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1β, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κβ1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes. PMID:23977054

  17. Differential gene expression and characterization of tissue-specific cDNA clones in oil palm using mRNA differential display.

    PubMed

    San, Cha Thye; Shah, Farida Habib

    2005-12-01

    The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein. PMID:16328884

  18. Cloning of the Aegiceras corniculatum class I chitinase gene (AcCHI I) and the response of AcCHI I mRNA expression to cadmium stress.

    PubMed

    Wang, Li-Ying; Wang, You-Shao; Cheng, Hao; Zhang, Jing-Ping; Yeok, Foong Swee

    2015-10-01

    Chitinases in terrestrial plants have been reported these are involved in heavy metal tolerance/detoxification. This is the first attempt to reveal chitinase gene (AcCHI I) and its function on metal detoxification in mangroves Aegiceras corniculatum. RT-PCR and RACE techniques were used to clone AcCHI I, while real-time quantitative PCR was employed to assess AcCHI I mRNA expressions in response to Cadmium (Cd). The deduced AcCHI I protein consists of 316 amino acids, including a signal peptide region, a chitin-binding domain (CBD) and a catalytic domain. Protein homology modeling was performed to identify potential features in AcCHI I. The CBD structure of AcCHI I might be critical for metal tolerance/homeostasis of the plant. Clear tissue-specific differences in AcCHI I expression were detected, with higher transcript levels detected in leaves. Results demonstrated that a short duration of Cd exposure (e.g., 3 days) promoted AcCHI I expression in roots. Upregulated expression was also detected in leaves under 10 mg/kg Cd concentration stress. The present study demonstrates that AcCHI I may play an important role in Cd tolerance/homeostasis in the plant. Further studies of the AcCHI I protein, gene overexpression, the promoter and upstream regulation will be necessary for clarifying the functions of AcCHI I. PMID:26044931

  19. Concordant Association of Insulin Degrading Enzyme Gene (IDE) Variants with IDE mRNA, Aß, and Alzheimer's Disease

    PubMed Central

    Ertekin-Taner, Nilufer; Ansari, Morad; Wilcox, Samantha L.; Kashino, Mariah R.; Ma, Li; Younkin, Linda H.; Younkin, Samuel G.; Younkin, Curtis S.; Dincman, Toros A.; Howard, Melissa E.; Howell, Chanley C.; Stanton, Chloe M.; Watson, Christopher M.; Crump, Michael; Vitart, Veronique; Hayward, Caroline; Hastie, Nicholas D.; Rudan, Igor; Campbell, Harry; Polasek, Ozren; Brown, Kristelle; Passmore, Peter; Craig, David; McGuinness, Bernadette; Todd, Stephen; Kehoe, Patrick G.; Mann, David M.; Smith, A. David; Beaumont, Helen; Warden, Donald; Holmes, Clive; Heun, Reinhard; Kölsch, Heike; Kalsheker, Noor; Pankratz, V. Shane; Dickson, Dennis W.; Graff-Radford, Neill R.; Petersen, Ronald C.; Wright, Alan F.; Younkin, Steven G.; Morgan, Kevin

    2010-01-01

    Background The insulin-degrading enzyme gene (IDE) is a strong functional and positional candidate for late onset Alzheimer's disease (LOAD). Methodology/Principal Findings We examined conserved regions of IDE and its 10 kb flanks in 269 AD cases and 252 controls thereby identifying 17 putative functional polymorphisms. These variants formed eleven haplotypes that were tagged with ten variants. Four of these showed significant association with IDE transcript levels in samples from 194 LOAD cerebella. The strongest, rs6583817, which has not previously been reported, showed unequivocal association (p = 1.5×10−8, fold-increase = 2.12,); the eleven haplotypes were also significantly associated with transcript levels (global p = 0.003). Using an in vitro dual luciferase reporter assay, we found that rs6583817 increases reporter gene expression in Be(2)-C (p = 0.006) and HepG2 (p = 0.02) cell lines. Furthermore, using data from a recent genome-wide association study of two Croatian isolated populations (n = 1,879), we identified a proxy for rs6583817 that associated significantly with decreased plasma Aβ40 levels (ß = −0.124, p = 0.011) and total measured plasma Aβ levels (b = −0.130, p = 0.009). Finally, rs6583817 was associated with decreased risk of LOAD in 3,891 AD cases and 3,605 controls. (OR = 0.87, p = 0.03), and the eleven IDE haplotypes (global p = 0.02) also showed significant association. Conclusions Thus, a previously unreported variant unequivocally associated with increased IDE expression was also associated with reduced plasma Aß40 and decreased LOAD susceptibility. Genetic association between LOAD and IDE has been difficult to replicate. Our findings suggest that targeted testing of expression SNPs (eSNPs) strongly associated with altered transcript levels in autopsy brain samples may be a powerful way to identify genetic associations with LOAD that would otherwise be difficult to detect

  20. Substrate stiffness regulates apoptosis and the mRNA expression of extracellular matrix regulatory genes in the rat annular cells.

    PubMed

    Zhang, Yue-Hui; Zhao, Chang-Qing; Jiang, Lei-Sheng; Dai, Li-Yang

    2011-03-01

    Cells are subjected to static tension of different magnitudes when cultured on substrates with different stiffnesses. It has long been recognized that mechanical stress is an important modulator of the intervertebral disc degeneration. Here we studied the influence of substrate stiffness on cell morphology, apoptosis and extracellular matrix (ECM) metabolism of the rat annulus fibrosus (AF) cells which are known to be mechanosensitive cells. Polyacrylamide gel substrates with three different stiffnesses were prepared by varying the concentration of acrylamide and bisacrylamide, and the elastic modulus of the different gel substrates were measured with atomic force microscopy (AFM). First-passage rat annular cells were cultured on soft, intermediate, rigid substrates or plastics for 24 or 48 h. The percentages of apoptotic cells were detected by flow cytometry and caspase-3 activity, and morphologic changes were visualized by Hoechst 33258 staining and F-actin staining. In addition, the expression of ECM genes (Col1α1, Col2α1, aggrecan, MMP-3, MMP-13 and ADAMTS-5) were analyzed by RT-PCR. The three different substrates had elastic moduli varying between 1±0.23 kPa (soft, 5% gel with 0.06% bis), 32±2.89 kPa (intermediate, 10% gel with 0.13% bis) and 63±3.45 kPa (rigid, 10% gel with 0.26% bis) with a thickness about 60-70 μm. Most of the rat AF cells appeared small and rounded, and lost most of their stress fibers when cultured on soft substrate. There was a significant increase in the percentage of apoptotic cells in the rat AF cells cultured on soft and intermediate substrates relative to those on plastic surface, with a parallel decrease in the area of cell spreading and nucleus. The AF cells grown on intermediate or rigid substrate had reduced expression of Col1α1, Col2α1 and aggrecan and enhanced expression of MMP-3, MMP-13, and ADAMTS-5 at 24h or 48 h, respectively, relative to those cultured on plastic surface. Conversely, we observed an up

  1. Changes of Antioxidant Function and the mRNA Expression Levels of Apoptosis Genes in Duck Ovaries Caused by Molybdenum or/and Cadmium.

    PubMed

    Cao, Huabin; Xia, Bing; Zhang, Mengmeng; Liao, Yilin; Yang, Zhi; Hu, Guoliang; Zhang, Caiying

    2016-06-01

    To investigate the effects of molybdenum (Mo) combined with cadmium (Cd) on the antioxidant function and the mRNA expression levels of apoptosis-related genes in duck ovaries, 60 healthy 11-old-day female ducks were treated with hexaammonium molybdate ([(NH4)6Mo7O24·4H2O]) or/and cadmium sulfate (3CdSO4·8H2O) at different doses on a daily basis for 120 days. On the 120th day, ten female birds in each group were euthanized, and the ovaries and blood were collected to determine the antioxidant indexes and the mRNA expression levels of Bak-1, Bcl-2, and caspase-3 in ovaries. In addition, ovary tissues were subjected to histopathological analysis with optical microscope. The total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity decreased significantly (P < 0.01) in treated groups comparing with control while the nitric oxide synthase (NOS) activity increased (P < 0.01) both in ovary tissue and serum. The Bak-1 and caspase-3 expressions were upregulated while the Bcl-2 was downgraded by Mo or/and Cd. Biomolecules were affected in all metal-treated groups, whereas combined-treated animals showed greater effects. What is more, pathological damage in Mo and Cd combination treated groups was more severe. The results from the present study indicated that Mo or/and Cd caused oxidative stress and apoptosis in duck ovaries. Combination of Mo and Cd showed additive or synergistic effect leading to apoptosis and oxidative stress, and the pathway might be the mitochondrial pathway. PMID:26446861

  2. Fibulin-2 (FBLN2): Human cDNA sequence, mRNA expression, and mapping of the gene on human and mouse chromosomes

    SciTech Connect

    Zhang, R.Z.; Pan, T.C.; Zhang, Z.Y.

    1994-07-15

    Fibulin-2 is a new extracellular matrix protein recently identified by characterizing mouse cDNA clones. Fibulin-2 mRNA is prominently expressed in mouse heart tissue and is present in low amounts in other tissues. In this study, the authors isolated and sequenced a 4.1-kb human fibulin-2 cDNA, which encoded a mature protein of 1157 amino acids preceded by a 27-residue signal sequence. The predicted polypeptide contains three consecutive anaphylatoxin-related segments (domain I) in its central region followed by 10 EGF-like repeats (domain II), 9 of which have a consensus sequence for calcium binding. The 408-residue N-terminal region consists of two separate subdomains, a cysteine-rich segment of 150 residues (Na subdomain) and a cysteine-free segment with a stretch of acidic amino acids (Nb subdomain). The 115-residue C-terminal segment (domain III) is similar to the C variant of fibulin-1. The amino acid sequences of the human and mouse fibulin-2 share {approximately}90% identity in domains Na, I, II, and III but only 62% identity in domain Nb. The human cDNA lacks an EGF-like repeat, which is alternatively spliced in the mouse cDNA clones, and a potential cell-binding Arg-Gly-Asp sequence found in the Nb domain of the mouse counterpart. Northern blot analysis of mRNA from various human tissues reveals an abundant 4.5-kb transcript in heart, placenta, and ovary tissue. The expression pattern differs from that of fibulin-1. The fibulin-2 gene was localized by in situ hybridization to the p24-p25 region of human chromosome 3 and to the band D-E of mouse chromosome 6. 27 refs., 5 figs.

  3. Effect of nutrition on plasma lipid profile and mRNA levels of ovarian genes involved in steroid hormone synthesis in Hu sheep during luteal phase.

    PubMed

    Ying, S J; Xiao, S H; Wang, C L; Zhong, B S; Zhang, G M; Wang, Z Y; He, D Y; Ding, X L; Xing, H J; Wang, F

    2013-11-01

    Ovarian steroid hormones regulate follicular growth and atresia. This study aims to determine whether key ovarian sterol-regulatory genes are differentially expressed in Hu sheep under different short-term nutritional regimens. Estrus was synchronized using intravaginal progestagen sponges. The ewes were assigned randomly to 3 groups. On d 6 to 12 of their estrous cycle, the control (CON) group received a maintenance diet (1.0×M), the supplemented (SUP) group received 1.5×M, and the restricted (R) group received 0.5×M. On d 7 to 12, blood samples were taken. The sheep were slaughtered at the end of the treatment, and their organs and ovaries were collected. The plasma concentrations of urea (P<0.01), total cholesterol (P<0.01), low-density lipoprotein cholesterol (P<0.01), NEFA (P<0.01), FSH (P<0.05), and estradiol (P<0.05) increased with decreasing dietary intake, whereas plasma triglyceride (P<0.01) and triiodothyronine (T3) concentrations decreased (P<0.05). The ewes in the R group had higher spleen weight and percentage of spleen to BW and lower liver and small intestine weights and percentage of liver/stomach to BW than the SUP group ewes (P<0.05). Nutritional restriction decreased the cytochrome p450 (CYP17A1) and estrogen receptor 1 (ESR1) mRNA expression (P<0.05) and increased the cytochrome p450 aromatase (CYP19A1) mRNA expression (P<0.05) in follicles>2.5 mm. Follicle size affected the mRNA expression of very low density lipoprotein receptor (VLDLR), estrogen receptor 2 (ESR2), FSH receptor (FSHR), CYP17A1, and CYP19A1 (P<0.05). In conclusion, we suggest that a potential mechanism by which short-term negative energy balance inhibits follicular growth may involve responses to disrupted reproductive hormone concentrations and influenced the intrafollicular expression of CYP17A1, CYP19A1, and ESR1. This result may be due to increased plasma urea and lipid concentrations. PMID:24045481

  4. A Genome-Wide mRNA Screen and Functional Analysis Reveal FOXO3 as a Candidate Gene for Chicken Growth

    PubMed Central

    Chen, Biao; Xu, Jiguo; He, Xiaomei; Xu, Haiping; Li, Guihuan; Du, Hongli; Nie, Qinghua; Zhang, Xiquan

    2015-01-01

    Chicken growth performance provides direct economic benefits to the poultry industry. However, the underlying genetic mechanisms are unclear. The objective of this study was to identify candidate genes associated with chicken growth and investigate their potential mechanisms. We used RNA-Seq to study the breast muscle transcriptome in high and low tails of Recessive White Rock (WRRh, WRRl) and Xinghua chickens (XHh, XHl). A total of 60, 23, 153 and 359 differentially expressed genes were detected in WRRh vs. WRRl, XHh vs. XHl, WRRh vs. XHh and WRRl vs. XHl, respectively. GO, KEGG pathway and gene network analyses showed that CEBPB, FBXO32, FOXO3 and MYOD1 played key roles in growth. The functions of FBXO32 and FOXO3 were validated. FBXO32 was predominantly expressed in leg muscle, heart and breast muscle. After decreased FBXO32 expression, growth-related genes such as PDK4, IGF2R and IGF2BP3 were significantly down-regulated (P < 0.05). FBXO32 was significantly (P < 0.05) associated with carcass and meat quality traits, but not growth traits. FOXO3 was predominantly expressed in breast and leg muscle. In both of these tissues, the FOXO3 mRNA level in XH was significantly higher than that in WRR chickens with normal body weight (P < 0.05). In DF-1 cells, siRNA knockdown of FOXO3 significantly (P < 0.01) inhibited the MYOD expression and significantly up-regulated (P < 0.01 or P < 0.05) the expression of growth-related genes including CEBPB, FBXO32, GH, GHR, IGF1R, IGF2R, IGF2BP1, IGF2BP3, INSR, PDK1 and PDK4. Moreover, 18 SNPs were identified in FOXO3. G66716193A was significantly (P < 0.05) associated with growth traits. The sites C66716002T, C66716195T and A66716179G were significantly (P < 0.05) associated with growth or carcass traits. These results demonstrated that FOXO3 is a candidate gene influencing chicken growth. Our observations provide new clues to understand the molecular basis of chicken growth. PMID:26366565

  5. Molecular cloning and mRNA expression of a hepcidin gene from the spinyhead croaker, Collichthys lucidus.

    PubMed

    Sang, C; Lin, Y; Jiang, K; Zhang, F; Song, W

    2015-01-01

    Antimicrobial peptides are important components that participate in host innate immune activities and play crucial roles in host defense against microbial invasion. Hepcidin is an antimicrobial peptide and iron-regulatory molecule that primarily functions in the liver. In the present study, we first obtained a full-length cDNA sequence of hepcidin and its corresponding genomic DNA sequence from Collichthys lucidus using RT-PCR and rapid amplification of cDNA ends (RACE), and then analyzed these sequences using bioinformatics software. The results showed that C. lucidus hepcidin (CL-hepc) possesses two introns and three exons in the genomic DNA, with a length of 816 bp. The open reading frame was 264 bp, encoding an 87 amino acid peptide, and with high similarity (88.89%) to 83416593 Larimichthys crocea (ABC18307) and relatively low similarity (47.73%) to 158358729 L. crocea (ABY84845.1). The pre-peptide contained a signal peptide (28 amino acids), a prodomain (34 amino acids), and a mature peptide (25 amino acids). The predicted 25 amino acid hepcidin mature peptide included 8 conserved cysteine residues. Quantitative real-time reverse transcription-PCR analysis revealed specific expression patterns of CL-hepc, with the highest expression observed in the liver, relatively low expression observed in the gill and spleen, and almost no expression detected in other tissues analyzed. In conclusion, we identified a hepcidin from C. lucidus that has common expression patterns with other hepcidins. However, as this hepcidin is inconsistent with two other hepcidins from L. crocea in terms of the phylogenetic tree, the presence of another hepcidin gene warrants further investigation. PMID:26662398

  6. Sexual maturation, serum steroid concentrations, and mRNA expression of IGF-1, luteinizing and progesterone hormone receptors and survivin gene in Japanese quail hens.

    PubMed

    Shit, N; Sastry, K V H; Singh, R P; Pandey, N K; Mohan, J

    2014-03-15

    In avian species, sexual maturation represents the evidence of start laying, which is a consequence of the development of ovarian follicles. These follicles are the functional reproductive unit whose maturation and viability critically depends on endocrine, paracrine, and autocrine factors beyond the signals from the central nervous system. The present study was undertaken to investigate the correlation of sexual maturity with tissue growth, mRNA expression of certain genes, and serum steroid concentrations in Japanese quail hens. To carry out the present study, a total of forty Japanese quail hens (5 weeks) were housed individually under uniform husbandry condition with ad libitum quail layer ration and water at 14-hour photo schedule. On sixth week onwards, four birds were sacrificed at each time on 1, 3, 7, 10, 13, 16, 19, 22, 25, and 28 days. Serum was extracted aseptically to analyze the gonadal steroid hormones (estrogen and progesterone) and corticosterone to investigate the liaison with sexual maturation of the species. Expression analyses of four genes i.e., insulin-like growth factor-1, luteinizing hormone receptor, progesterone receptor, and survivin were carried out in the three largest ovarian yellow follicles. A significant (P < 0.05) increase in body weight gain and oviduct weight was recorded during the phase of sexual maturation. Smaller follicles revealed higher insulin-like growth factor-1 and survivin gene expression, whereas the reverse result was manifested in both the luteinizing and progesterone hormone receptors. In biochemical study, the gonadal steroids (estrogen and progesterone) were recorded higher at the first half of the experiment when a gradual decrease in corticosterone concentration was confirmed from the very beginning of this study. This result substantiated that sexual maturation in Japanese quail may be completed by the time of 8 weeks after its birth in support of the analyzed information studied in the current investigation

  7. The effect of 1,25 dihydroxyvitamin D3 treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles

    PubMed Central

    DeWitt, Marsha; Johnson, Robert L.; Snyder, Paul; Fleet, James C.

    2015-01-01

    In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)2D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)2D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions ofmice with colon-specific deletion of both APC gene alleles (CAC; APCΔ580/Δ580). At four weeks of age, CAC; APCΔ580/Δ580 and control mice were injected with vehicle or 1,25(OH)2D (1 μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APCΔ580/Δ580 mice. 1,25(OH)2D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APCΔ580/Δ580 and control mice indicating the treatments activated the VDR. However, 1,25(OH)2D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APCΔ580/Δ580 or control mice. VDR mRNA and protein levels were lower (−65% and −90%) in the colon of CAC; APCΔ580/Δ580 mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of Transient Receptor Potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APCΔ580/Δ580 mice. Our data show that short term exposure to 1,25(OH)2D does not suppress colonic β-catenin signaling in vivo. PMID:25597951

  8. Gene expression profiling by mRNA sequencing reveals increased expression of immune/inflammation-related genes in the hippocampus of individuals with schizophrenia

    PubMed Central

    Hwang, Y; Kim, J; Shin, J-Y; Kim, J-II; Seo, J-S; Webster, M J; Lee, D; Kim, S

    2013-01-01

    Whole-genome expression profiling in postmortem brain tissue has recently provided insight into the pathophysiology of schizophrenia. Previous microarray and RNA-Seq studies identified several biological processes including synaptic function, mitochondrial function and immune/inflammation response as altered in the cortex of subjects with schizophrenia. Now using RNA-Seq data from the hippocampus, we have identified 144 differentially expressed genes in schizophrenia cases as compared with unaffected controls. Immune/inflammation response was the main biological process over-represented in these genes. The upregulation of several of these genes, IFITM1, IFITM2, IFITM3, APOL1 (Apolipoprotein L1), ADORA2A (adenosine receptor 2A), IGFBP4 and CD163 were validated in the schizophrenia subjects using data from the SNCID database and with quantitative RT-PCR. We identified a co-expression module associated with schizophrenia that includes the majority of differentially expressed genes related to immune/inflammation response as well as with the density of parvalbumin-containing neurons in the hippocampus. The results indicate that abnormal immune/inflammation response in the hippocampus may underlie the pathophysiology of schizophrenia and may be associated with abnormalities in the parvalbumin-containing neurons that lead to the cognitive deficits of the disease. PMID:24169640

  9. Altered mRNA expression of the Rb and p16 tumor suppressor genes and of CDK4 in transitional cell carcinomas of the urinary bladder associated with tumor progression.

    PubMed

    Quentin, Thomas; Henke, Christian; Korabiowska, Monika; Schlott, Thilo; Zimmerman, Britt; Kunze, Ekkehard

    2004-01-01

    Based on the concept that tumor suppressor genes are involved in the pathogenesis of urinary bladder carcinogenesis, we analysed the mRNA expression of the retinoblastoma (Rb) and p16 (CDKN2, INK4A, MTS1) genes as well as of the proto-oncogene cyclin D-dependent kinase 4 (CDK4) in 71 transitional cell carcinomas (TCC) of the urinary bladder in relation to the tumor grades and stages, and with reference to certain lifestyle and occupational risk factors. Using real-time quantitative reverse transcription-polymerase chain reaction, high-stage muscle invasive TCC expressed the Rb, p16 and CDK4 mRNA at lower levels than low-stage superficial cancers, indicating down-regulation to be linked with tumor progression. The drop of the expression in the group of grade 2 TCC when invading the muscle layer compared to grade 2 carcinomas with a superficial pattern of growth is considered to represent a key event in promoting urothelial carcinogenesis in this subset of carcinomas. The protein expression of the Rb gene evaluated by immunohistochemistry proved to be closely related to the tumor grades and stages as well as to the mRNA expression, high-grade and high-stage TCC disclosing a lower rate of positive immunoreactivity than low-grade and low-stage carcinomas. The p16 protein product was expressed at a lower level in grade 3 than in grade 1 TCC, but there was no correlation with the tumor stages or the mRNA expression. TCC with loss of heterozygosity (LOH) at the INK4A region showed a decreased expression of p16 mRNA compared to those without an allelic loss. Tobacco smoke was not identified to substantially modulate the Rb/p16/CDK4 pathways, except for a ten-fold elevated mRNA expression of the p16 gene in TCC of light compared to heavy smokers. Heavy coffee consumption was associated with a reduced expression of CDK4 mRNA. Among occupational exposures, TCC of patients in contact with stone dust, paints and lacquer, plastics, wood and wood preservers and chemical solvents

  10. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene.

    PubMed

    Clancy, Mary J; Shambaugh, Mary Eileen; Timpte, Candace S; Bokar, Joseph A

    2002-10-15

    N6-methyladenosine (m6A) is present at internal sites in mRNA isolated from all higher eukaryotes, but has not previously been detected in the mRNA of the yeast Saccharomyces cerevisiae. This nucleoside modification occurs only in a sequence- specific context that appears to be conserved across diverse species. The function of this modification is not fully established, but there is some indirect evidence that m6A may play a role in the efficiency of mRNA splicing, transport or translation. The S.cerevisiae gene IME4, which is important for induction of sporulation, is very similar to the human gene MT-A70, which has been shown to be a critical subunit of the human mRNA [N6-adenosine]-methyltransferase. This observation led to the hypothesis that yeast sporulation may be dependent upon methylation of yeast mRNA, mediated by Ime4p. In this study we show that induction of sporulation leads to the appearance of low levels of m6A in yeast mRNA and that this modification requires IME4. Moreover, single amino acid substitutions in the putative catalytic residues of Ime4p lead to severe sporulation defects in a strain whose sporulation ability is completely dependent on this protein. Collectively, these data suggest very strongly that the activation of sporulation by Ime4p is the result of its proposed methyltransferase activity and provide the most direct evidence to date of a physiologic role of m6A in a gene regulatory pathway. PMID:12384598

  11. Fructose containing sugars modulate mRNA of lipogenic genes ACC and FAS and protein levels of transcription factors ChREBP and SREBP1c with no effect on body weight or liver fat.

    PubMed

    Janevski, Mile; Ratnayake, Sunil; Siljanovski, Svetlana; McGlynn, Maree A; Cameron-Smith, David; Lewandowski, Paul

    2012-02-01

    The aim of this study was to determine the effects of high-glucose, high-fructose and high-sucrose diets on weight gain, liver lipid metabolism and gene expression of proteins involved with hepatic fat metabolism. Rats were fed a diet containing either 60% glucose, 60% fructose, 60% sucrose, or a standard chow for 28 days. Results indicated that high-fructose and high-sucrose diets were associated with higher mRNA levels of gene transcripts involved with fat synthesis; ACC, FAS and ChREBP, with no change in SREBP-1C mRNA. The protein level of ChREBP and SREBP1c was similar in liver homogenates from all groups, but were higher in nuclear fractions from the liver of high-fructose and high-sucrose fed rats. The mRNA level of gene transcripts involved with fat oxidation was the same in all three diets, whilst a high-fructose diet was associated with greater amount of mRNA of the fat transporter CD36. Despite the changes in mRNA of lipogenic proteins, the body weight of animals from each group was the same and the livers from rats fed high-fructose and high-sucrose diets did not contain more fat than control diet livers. In conclusion, changing the composition of the principal monosaccharide in the diet to a fructose containing sugar elicits changes in the level of hepatic mRNA of lipogenic and fat transport proteins and protein levels of their transcriptional regulators; however this is not associated with any changes in body weight or liver fat content. PMID:22159273

  12. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  13. Analysis of anomalous CD44 gene expression in human breast, bladder, and colon cancer and correlation of observed mRNA and protein isoforms.

    PubMed Central

    Woodman, A. C.; Sugiyama, M.; Yoshida, K.; Sugino, T.; Borgya, A.; Goodison, S.; Matsumura, Y.; Tarin, D.

    1996-01-01

    Many studies have now demonstrated disorganized overexpression of the CD44 gene in various types of human malignant tumors, and this abnormality has emerged as an interesting candidate marker for early cancer diagnosis. The purpose of this work was to analyze and compare the patterns of transcription and translation of this gene in human breast (ZR75-1; MDAMB-435 clone 4A4) and colon (HT29) tumor cell lines and in tumors of the breast, bladder, and colon, with the aim of identifying the most suitable analyte for diagnostic purposes. Transcription was studied by reverse transcription-polymerase chain reaction using CD44-specific primers and probes complementary to exons in the standard (exons 3 to 5 and 16 to 18) and variably expressed regions of this gene (exons 7, 8, 10, 11, and 15). Translation was investigated by Western blot analysis and immunohistochemistry using monoclonal antibodies specific to the standard form of CD44 and to the products of the same variant exons. Southern blot hybridization analysis of the reverse transcription-polymerase chain reaction products showed a large number of CD44 transcripts in tumor cells. Direct comparison of these Southern blots with Western blots on matched tumor-cell-line extracts indicated that most of the diverse mRNA isoforms did not detectably translate into proteins. However, immunohistochemistry of normal and malignant breast (n = 17 and 23, respectively), bladder (n = 5 and 19), and colon (n = 19 and 19) tissue specimens showed increased staining of CD44 standard and CD44 variant proteins in the carcinoma cells. Combination of this information with the data from reverse transcription-polymerase chain reaction and Western blot analysis indicates that the overexpression at the protein level involves only a minority of the aberrant RNA transcripts. We conclude that the development of methods for the accurate quantitation of over-abundant CD44 RNA species in clinical samples offers the most promising approach to

  14. Maternal separation enhances conditioned fear and decreases the mRNA levels of the neurotensin receptor 1 gene with hypermethylation of this gene in the rat amygdala.

    PubMed

    Toda, Hiroyuki; Boku, Shuken; Nakagawa, Shin; Inoue, Takeshi; Kato, Akiko; Takamura, Naoki; Song, Ning; Nibuya, Masashi; Koyama, Tsukasa; Kusumi, Ichiro

    2014-01-01

    Stress during postnatal development is associated with an increased risk for depression, anxiety disorders, and substance abuse later in life, almost as if mental illness is able to be programed by early life stressors. Recent studies suggest that such "programmed" effects can be caused by epigenetic regulation. With respect to conditioned fear, previous studies have indicated that early life stress influences its development in adulthood, whereas no potential role of epigenetic regulation has been reported. Neurotensin (NTS) is an endogenous neuropeptide that has receptors densely located in the amygdala and hippocampus. Recently, NTS systems have constituted an emerging target for the treatment of anxiety. The aim of the present work is to clarify whether the NTS system is involved in the disturbance of conditioned fear in rats stressed by maternal separation (MS). The results showed that MS enhanced freezing behaviors in fear-conditioned stress and reduced the gene expression of NTS receptor (NTSR) 1 but not of NTS or NTSR2 in the amygdalas of adult rats. The microinjection of a NTSR1 antagonist into the amygdala increased the percentage of freezing in conditioned fear, whereas the microinjection of NTSR1 agonist decreased freezing. These results suggest that NTSR1 in the amygdala may play a role in the effects of MS on conditioned fear stress in adult rats. Moreover, MS increased DNA methylation in the promoter region of NTSR1 in the amygdala. Taken together, MS may leave epigenetic marks in the NTSR1 gene in the amygdala, which may enhance conditioned fear in adulthood. The MS-induced alternations of DNA methylation in the promoter region of NTSR1 in the amygdala may be associated with vulnerability to the development of anxiety disorders and depression in adulthood. PMID:24831231

  15. Expression characteristics of neutrophil and mononuclear-phagocyte related genes mRNA in the stable angina pectoris and acute myocardial infarction stages of coronary artery disease

    PubMed Central

    Li, Chuan-Rong; Wang, Le-Min; Gong, Zhu; Jiang, Jin-Fa; Duan, Qiang-Lin; Yan, Wen-Wen; Liu, Xiao-Hui

    2015-01-01

    Objective To investigate expression differences of neutrophil and mononuclear phagocyte related gene mRNAs among acute myocardial infarction (AMI), stable angina (SA) and control groups, and then discuss their expression characteristics in the stable angina pectoris (SAP) and AMI stages of coronary artery disease (CAD). Methods Whole Human Genome Oligo Microarrays were applied to assess the differential expression characteristics of neutrophil and mononuclear phagocyte related mRNAs in patients with AMI (n = 20), SA (n = 20) and controls (n = 20). Results (1) Almost all colony-stimulating factors (CSF) and their receptors related mRNAs was up-regulated in AMI and SA groups compared with the control group, and the expression of granulocyte-macrophage colony stimulating factor receptor (GM-CSFR) and granulocyte colony stimulating factor receptor (G-CSFR) mRNAs in the AMI group was significantly up-regulated compared with the other two groups (P < 0.01). (2) The expression of mRNAs related to monocyte chemoattractant protein-1 (MCP-1), CCR2 (MCP-1 receptor) and CXCR2 (IL-8 receptor) was significantly up-regulated (P < 0.01) in AMI group compared with SA and control groups. IL-8 mRNA expression in the AMI group was clearly higher than the controls (P < 0.05). (3) All mRNAs expression related to opsonic receptors (IgG FcR and C3bR/C4bR) was significantly up-regulated in AMI group compared with SA and control group (P < 0.01), and the SA group showed an upward trend compared with controls. (4) Most pattern recognition receptor (PRR)-related mRNAs expression was up-regulated in AMI group compared with SA and control groups. Most toll-like receptor (TLR) mRNAs expression was significantly up-regulated (P < 0.01) than the SA and control groups; macrophage scavenger receptor (MSR) mRNA was significantly up-regulated in AMI group compared with the control group (P < 0.01), and the SA group showed an upward trend compared with the controls. Conclusions The expression

  16. Intrauterine growth restriction in neonatal piglets affects small intestinal mucosal permeability and mRNA expression of redox-sensitive genes.

    PubMed

    Wang, Wei; Degroote, Jeroen; Van Ginneken, Chris; Van Poucke, Mario; Vergauwen, Hans; Dam, Thi Minh Tho; Vanrompay, Daisy; Peelman, Luc J; De Smet, Stefaan; Michiels, Joris

    2016-02-01

    Neonates with intrauterine growth restriction (IUGR) show lower efficiency of nutrient utilization compared to normal birth weight (NBW) newborns. This study was conducted using neonatal piglets as a model to test the hypothesis that IUGR affects the intestinal barrier function, intestinal structure, and antioxidant system development during the suckling period. The small intestinal mucosae were obtained from IUGR and NBW littermates in the suckling period (d 0, 3, 8, and 19 postnatal). The epithelial barrier function was assessed by FITC-dextran 4 (FD4) and horseradish peroxidase (HRP) fluxes across the epithelium, histomorphologic measurements, and expression of tight-junction proteins. Redox status represented by the glutathione disulfide/glutathione ratio and malondialdehyde concentrations was determined, whereas mRNA expressions of some redox-sensitive proteins were quantified. Results showed that IUGR piglets exhibited a 2-fold higher intestinal permeability in the proximal small intestine on d 0 (P < 0.05), and this difference between IUGR and NBW piglets was widened to 3 and 4 times for FD4 and HRP, respectively (P < 0.05), on d 3. In accordance, expression of occludin was down-regulated at the transcriptional level in IUGR piglets at d 0 and 19 (P < 0.01). Furthermore, the transcription of heme oxygenase 1, catalase, and thioredoxin reductase genes was down-regulated in IUGR piglets, mainly on postnatal d 0 and 19 (P < 0.01). It appears that IUGR subjects have a lower capacity to mount an antioxidant response in the early postnatal period. Collectively, these results add to our understanding of the mechanisms responsible for intestinal dysfunction in IUGR neonates. PMID:26514167

  17. The human and mouse homologs of the yeat RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues

    SciTech Connect

    Shen, Z.; Chen, D.J.; Denison, K.

    1995-01-01

    The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an {approximately}1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of {approximately}0.85 kb was detectable in mouse testis. 40 refs., 7 figs., 1 tab.

  18. Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time.

    PubMed

    Peng, Zhiyong; Young, Brandon; Baird, Alison E; Soper, Steven A

    2013-08-20

    Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for in vitro diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with online single-molecule detection following the LDR. As demonstrators of the assay's utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10-10,000 copies) with an R(2) = 0.9995 for RT-LDR/spFRET and R(2) = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a two thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time

  19. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs

    PubMed Central

    Men, X. M.; Deng, B.; Tao, X.; Qi, K. K.; Xu, Z. W.

    2016-01-01

    The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics. PMID:26949945

  20. Explore the dynamic alternation of gene PLAC4 mRNA expression levels in maternal plasma in second trimester for nonivasive detection of trisomy 21

    PubMed Central

    Sun, Hai-Yan; Chen, Dao-Zhen; Lu, Mu-Dan; Tang, Ye; Xiao, Jian-Pin

    2015-01-01

    Objective Noninvasive prenatal detection of trisomy 21 (T21) has been achieved by measuring the ratio of two alleles of a single nucleotide polymorphism in circulating placenta specific 4 (PLAC4) mRNA in maternal plasma with a few assays in recent years. Our research is to explore the variations of PLAC4 mRNA expression level in maternal plasma with normal pregnancies in second trimester, which can provide pregnant women deeper insights with suitable detection period for the non-invasive prenatal detection of T21. Methods We measured a serial plasma PLAC4 mRNA concentrations weekly from the same 25 singleton normal pregnant women. We recruited maternal plasma samples from 45 singleton pregnant women, comprising of 25 euploid pregnancies (control group; range, 17 to 21 weeks) and 20 T21 pregnancies (T21 group; range, 19 to 24 weeks). With the application of reverse transcription polymerase chain reaction, we achieved an insight of PLAC4 mRNA expression levels in maternal plasma during second trimester with euploid pregnancies. Results Among the control group, the levels of PLAC4 mRNA expression in the gestation of 17 to 18 weeks were significantly less than those in the gestation of 18 to 21 weeks (P<0.05). The average PLAC4 mRNA concentration of the normal pregnant women was not higher than that of the T21 group (P>0.05). Conclusion The PLAC4 mRNA showed a higher level of expression in the gestation of 18 to 21 weeks with an euploid pregnancy of pregnant women. We also found that there was no significant difference in plasma PLAC4 mRNA concentration between the normal and the T21 pregnancies in second trimester. PMID:26217595

  1. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs.

    PubMed

    Men, X M; Deng, B; Tao, X; Qi, K K; Xu, Z W

    2016-04-01

    The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics. PMID:26949945

  2. [Determination of the amount of YB-1 gene mRNA in the breast tumor tissues to predict the course of disease].

    PubMed

    Gens, G P; Moiseeva, N I; Stromskaia, T P; Rybalkina, E Iu; Vaĭman, A V; Stavrovskaia, A A

    2010-02-01

    The purpose of the study was to develop a test of the multifunctional protein YB-1 in the intraoperative biopsy specimen to predict the course of breast cancer (BC). Its tasks were to use of real-time reverse-transcription polymerase chain reaction (RT-RCR) to substantiate the data previously obtained by semiquantitative RT-PCR and to clarify whether there was a correlation between the amount of YB-1 mRNA in the BC tissue and the status of steroid hormone receptors of these tumors. The determination of the tumor amount of YB-1 mRNA was shown to predict the course of BC: a statistically significant correlation was found between the higher content of YB-1 mRNA and the aggressive course of BC--the emergence of distant metastases. Comparing the content of YB-1 mRNA and the hormonal status of a tumor (the number of estrogen and progesterone receptors) revealed no correlations. The findings indicate that the determination of YB-1 mRNA by both real-time RT-PCR and semiquantitative RT-PCR may be used to predict BC metastases in distant organs. PMID:20397575

  3. mRNA expression levels and genetic status of genes involved in the EGFR and NF-κB pathways in metastatic non-small-cell lung cancer patients

    PubMed Central

    2011-01-01

    Background Metastatic non-small-cell lung cancer (NSCLC) has a dismal prognosis. EGFR is overexpressed or mutated in a large proportion of cases. Downstream components of the EGFR pathway and crosstalk with the NF-κB pathway have not been examined at the clinical level. We explored the prognostic significance of the mRNA expression of nine genes in the EGFR and NF-κB pathways and of BRCA1 and RAP80 in patients in whom EGFR and K-ras gene status had previously been determined. In addition, NFKBIA and DUSP22 gene status was also determined. Methods mRNA expression of the eleven genes was determined by QPCR in 60 metastatic NSCLC patients and in nine lung cancer cell lines. Exon 3 of NFKBIA and exon 6 of DUSP22 were analyzed by direct sequencing. Results were correlated with outcome to platinum-based chemotherapy in patients with wild-type EGFR and to erlotinib in those with EGFR mutations. Results BRCA1 mRNA expression was correlated with EZH2, AEG-1, Musashi-2, CYLD and TRAF6 expression. In patients with low levels of both BRCA1 and AEG-1, PFS was 13.02 months, compared to 5.4 months in those with high levels of both genes and 7.7 months for those with other combinations (P = 0.025). The multivariate analysis for PFS confirmed the prognostic role of high BRCA1/AEG-1 expression (HR, 3.1; P = 0.01). Neither NFKBIA nor DUSP22 mutations were found in any of the tumour samples or cell lines. Conclusions The present study provides a better understanding of the behaviour of metastatic NSCLC and identifies the combination of BRCA1 and AEG-1 expression as a potential prognostic model. PMID:21951562

  4. The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA.

    PubMed

    Xu, Fenghao; Morin, Charles; Mitchell, Grant; Ackerley, Cameron; Robinson, Brian H

    2004-08-15

    Leigh syndrome French Canadian (LSFC) is a variant of cytochrome oxidase deficiency found in Québec and caused by mutations in the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene. Northern blots showed that the LRPPRC mRNA levels seen in skeletal muscle>heart>placenta>kidney>liver>lung=brain were proportionally almost opposite in strength to the severity of the enzymic cytochrome oxidase defect. The levels of COX (cytochrome c oxidase) I and COX III mRNA visible on Northern blots were reduced in LSFC patients due to the common (A354V, Ala354-->Val) founder mutation. The amount of LRPPRC protein found in both fibroblast and liver mitochondria from LSFC patients was consistently reduced to <30% of control levels. Import of [(35)S]methionine LRPPRC into rat liver mitochondria was slower for the mutant (A354V) protein. A titre of LRPPRC protein was also found in nuclear fractions that could not be easily accounted for by mitochondrial contamination. [35S]Methionine labelling of mitochondrial translation products showed that the translation of COX I, and perhaps COX III, was specifically reduced in the presence of the mutation. These results suggest that the gene product of LRPPRC, like PET 309p, has a role in the translation or stability of the mRNA for mitochondrially encoded COX subunits. A more diffuse distribution of LRPPRC in LSFC cells compared with controls was evident when viewed by immunofluorescence microscopy, with less LRPPRC present in peripheral mitochondria. PMID:15139850

  5. Environmental enrichment attenuates the age-related decline in the mRNA expression of steroidogenic enzymes and reduces the methylation state of the steroid 5α-reductase type 1 gene in the rat hippocampus.

    PubMed

    Rossetti, María F; Varayoud, Jorgelina; Moreno-Piovano, Guillermo S; Luque, Enrique H; Ramos, Jorge G

    2015-09-01

    We analyzed the effects of aging and environmental enrichment on the mRNA expression and DNA methylation state of steroidogenic enzymes in the hippocampus. The effects of aging were evaluated by comparing young adult (90-day-old) and middle-aged (450-day-old) female Wistar rats. To elucidate the effects of environmental enrichment, a subgroup of middle-aged rats exposed to sensory and social stimulation for 105 days was compared to rats housed under standard laboratory conditions. Aging decreased the transcription of neurosteroidogenic-related genes and increased the promoter methylation state of cytochrome P450 side chain cleavage, 3α-hydroxysteroid dehydrogenase (3α-HSD) and 5α-reductase-1. Exposure of middle-aged rats to environmental enrichment increased mRNA levels of 5α-reductase-1, 3α-HSD and cytochrome P450 17α-hydroxylase/c17,20-lyase and decreased the methylation state of the 5α-reductase-1 gene. Thus, sensory and social stimulation attenuate the age-related decline in the mRNA expression of hippocampal steroidogenic enzymes. Epigenetic mechanisms associated with differential promoter methylation could be involved. PMID:26021641

  6. Two CYP3A-like genes in the marine mussel Mytilus edulis: mRNA expression modulation following short-term exposure to endocrine disruptors.

    PubMed

    Cubero-Leon, Elena; Puinean, A Mirel; Labadie, Pierre; Ciocan, Corina; Itoh, Naoki; Kishida, Mitsuyo; Osada, Makoto; Minier, Christophe; Hill, Elizabeth M; Rotchell, Jeanette M

    2012-03-01

    Members of the vertebrate CYP3A subfamily are involved in the metabolism of steroids and a wide range of xenobiotics. In this study two CYP3A-like mRNAs have been isolated from the mussel (Mytilus edulis), and their seasonal expression profile and modulation by estrogens examined. Sexual dimorphism of CYP3A-like mRNA expression was not observed in mussel gonads of individuals collected throughout a year. Nevertheless, natural variation in gonadal CYP3A-like mRNA expression was observed, with highest levels of CYP3A isoform1 and lowest levels of CYP3A isoform2 mRNA during the maturation and spawning season. Exposure to a 10% sewage treatment works extract did not result in any significant changes in mRNA expression of CYP3A-like. In contrast, exposure to E2 (200 ng/L) and TBT (100 ng/L) significantly down-regulated the expression of CYP3A-like isoform1 but not CYP3A-like isoform2 suggesting differential regulation. PMID:22189070

  7. Molecular analysis of methylmalonic acidemia: Identification of novel mutations in the methylmalonyl-CoA mutase gene with decreased level of mutant mRNA

    SciTech Connect

    Ogasawara, M.; Matsubara, Y.; Mikami, H.; Narisawa, K.

    1994-09-01

    Deficiency of methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, which is inherited as an autosomal recessive trait and clinically characterized by metabolic ketoacidosis. Previous studies of Caucasian and African American patients identified seven MCM mutations, and we also detected four missense substitutions (Ala197Thr, Val368Asp, Arg369His and Val669Glu). However, mutations with decreased level of MCM mRNA, which accounts for at least 25% of mutations among Caucasian patients, have not been reported. Our study on eight Japanese patients indicated that 13 of 16 mutant alleles (81%) showed decreased level of MCM mRNA, suggesting that these {open_quotes}low message{close_quotes} alleles are likely to be common contributors to MCM deficiency. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of these mutant mRNAs was less than 1% controls. We were able to amplify such mutant mRNAs by nested PCR and directly determine the primary structure. Sequence analysis revealed three novel mutations: a G-to-T substitution at nucleotide position 425, a 2 bp deletion at nt 769 and 770, and a G-to-T substitution at nt 326. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The analysis of 17 Japanese patients revealed the presence of G425T in 7 alleles (21%), suggesting a relatively high incidence of the mutation among Japanese patients. This observation is in sharp contrast to previous reports describing diverse heterogeneity of MCM mutations among Caucasians. Our report is the first to identify MCM mutations that decrease the stability of MCM mRNA. Amplification of trace amount of mRNA followed by sequencing analysis may provide useful tool for identifying such mutations.

  8. Analysis of mRNA expression for genes associated with regulatory T lymphocytes (CD25, FoxP3, CTLA4, and IDO) after experimental infection with bovine viral diarrhea virus of low or high virulence in beef calves.

    PubMed

    Palomares, Roberto A; Hurley, David J; Woolums, Amelia R; Parrish, Jacqueline E; Brock, Kenny V

    2014-12-01

    Immunosuppression caused by bovine viral diarrhea virus (BVDV) has been associated with lymphocyte depletion, leukopenia and impairment of leukocyte function; however, no work has been done on the relationship between BVDV and regulatory T lymphocytes (Tregs). The objective of this study was to compare the mRNA expression of genes associated with Tregs (CD25, FoxP3, CTLA4, and IDO), after experimental infection of beef calves with low (LV) or high (HV) virulence BVDV. Thirty BVDV-naïve calves were randomly assigned to three groups. Calves were intra-nasally inoculated with LV (n=10, strain SD-1) or HV (n=10, strain 1373) BVDV or BVDV-free cell culture medium (control, n=10). Quantitative RT-PCR was used to determine the expression of target genes in tracheo-bronchial lymph nodes and spleen on day 5 post-infection. The mRNA expression of CD25 was up-regulated in tracheo-bronchial lymph nodes of LV (P<0.05), but not in HV compared to the control group. The expression of FoxP3 and CTLA4 was not increased in tracheo-bronchial lymph nodes of either of the BVDV-inoculated groups. A dramatic up-regulation of IDO mRNA was observed in tracheo-bronchial lymph nodes of LV (P<0.05), but not HV compared to the control calves. In conclusion, experimental infection with BVDV did not provide evidence of Treg activation based on expression of FoxP3 and CTL4. Differential expression of CD25 and IDO mRNA on day 5 post-infection with HV or LV BVDV might reflect temporal differences in transcription occurring during the immune response elicited by these viral strains, or differences in viral infectivity of the host cells. PMID:25456194

  9. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA

    PubMed Central

    Munroe, Stephen H.; Morales, Christopher H.; Duyck, Tessa H.; Waters, Paul D.

    2015-01-01

    The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3’ end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3’splice site of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

  10. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA.

    PubMed

    Munroe, Stephen H; Morales, Christopher H; Duyck, Tessa H; Waters, Paul D

    2015-01-01

    The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3' end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3'splice site of TRα2 mRNA and antisense to the 3'UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

  11. Luteinizing hormone and follicle-stimulating hormone receptors and their transcribed genes (mRNA) are present in the lower urinary tract of intact male and female dogs.

    PubMed

    Ponglowhapan, S; Church, D B; Scaramuzzi, R J; Khalid, M

    2007-01-15

    In dogs, one of the side effects of neutering is the development of urinary incontinence. The relationship between neutering and urinary incontinence caused by acquired urethral sphincter mechanism incompetence (USMI) has been reported. Recently, GnRH analogue treatment that suppresses elevated plasma gonadotrophin concentrations post-spaying has been successfully used in incontinent bitches. These data and the fact that non-gonadal tissues may contain receptors for LH (LHR) and FSH (FSHR) suggest that there might be a functional relationship between gonadotrophins and the lower urinary tract in dogs. This study aimed to investigate the presence of LHR and FSHR in the lower urinary tract of intact male and female dogs. Four regions of the lower urinary tract, i.e. (i) body of the bladder, (ii) neck of the bladder, (iii) proximal urethra and (iv) distal urethra were collected from 10 healthy dogs (5 males and 5 anoestrous females). In situ hybridization and immunohistochemistry were performed to characterise the presence of receptor mRNA and receptor protein. Staining was rated semi-quantitatively, incorporating both the distribution and intensity of specific staining. The distribution of receptor expression in different tissue layers (epithelium, subepithelial stroma and muscle) in each region was statistically analyzed. Luteinizing hormone receptor and FSHR mRNA and protein were present in all four regions and in three tissue layers of males and females. Irrespective of region and layer, female dogs expressed significantly higher expression for LHR mRNA (P<0.001), LHR protein (P<0.05) and FSHR protein (P<0.001). The expression of LHR and FSHR mRNA and protein was not uniform and depended on region, tissue layer and gender. The expression of LHR mRNA was higher in the bladder, compared to the urethra (P<0.05). The FSHR mRNA significantly increased from the bladder to the urethra. Protein expression for LHR and FSHR was highest in the proximal urethra (P<0.05). The

  12. Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

    PubMed

    Yang, Zhiyong; Edenberg, Howard J; Davis, Ronald L

    2005-01-01

    To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes. PMID:16204451

  13. The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression

    PubMed Central

    Lamana, Amalia; López-Santalla, Mercedes; Castillo-González, Raquel; Ortiz, Ana María; Martín, Javier; García-Vicuña, Rosario; González-Álvaro, Isidoro

    2015-01-01

    Objective The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. Methods We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. Results After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. Conclusion Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway. PMID:26569609

  14. Cyclin-dependent kinase 7 controls mRNA synthesis by affecting stability of preinitiation complexes, leading to altered gene expression, cell cycle progression, and survival of tumor cells.

    PubMed

    Kelso, Timothy W R; Baumgart, Karen; Eickhoff, Jan; Albert, Thomas; Antrecht, Claudia; Lemcke, Sarah; Klebl, Bert; Meisterernst, Michael

    2014-10-01

    Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis. PMID:25047832

  15. Cyclin-Dependent Kinase 7 Controls mRNA Synthesis by Affecting Stability of Preinitiation Complexes, Leading to Altered Gene Expression, Cell Cycle Progression, and Survival of Tumor Cells

    PubMed Central

    Kelso, Timothy W. R.; Baumgart, Karen; Eickhoff, Jan; Albert, Thomas; Antrecht, Claudia; Lemcke, Sarah; Klebl, Bert

    2014-01-01

    Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis. PMID:25047832

  16. The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA.

    PubMed Central

    von Lindern, M; Fornerod, M; van Baal, S; Jaegle, M; de Wit, T; Buijs, A; Grosveld, G

    1992-01-01

    The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present in t(6;9) AML, encodes an invariable dek-can transcript. Sequence analysis of the dek-can cDNA showed that dek and can are merged without disruption of the original open reading frames and therefore the fusion mRNA encodes a chimeric DEK-CAN protein of 165 kDa. The predicted DEK and CAN proteins have molecular masses of 43 and 220 kDa, respectively. Sequence comparison with the EMBL data base failed to show consistent homology with any known protein sequences. Images PMID:1549122

  17. Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, Scophthalmus maximus.

    PubMed

    Urbatzka, R; Galante-Oliveira, S; Rocha, E; Lobo-da-Cunha, A; Castro, L F C; Cunha, I

    2015-07-01

    Fibrates and other lipid regulator drugs are widespread in the aquatic environment including estuaries and coastal zones, but little is known on their chronic effects on non-target organisms as marine fish. In the present study, turbot juveniles were exposed to the PPARα model agonist WY-14,643 for 21 days by repeated injections at the concentrations of 5mg/kg (lo-WY) and 50mg/kg (hi-WY), and samples taken after 7 and 21 days. Enzyme activity and mRNA expression of palmitoyl-CoA oxidase and catalase in the liver were analyzed as first response, which validated the experiment by demonstrating interactions with the peroxisomal fatty acid oxidation and oxidative stress pathways in the hi-WY treatment. In order to get mechanistic insights, alterations of plasma lipids (free cholesterol, FC; HDL associated cholesterol, C-HDL; triglycerides, TG; non-esterified fatty acids, NEFA) and hepatic mRNA expression of 17 genes involved in fatty acid and lipid metabolism were studied. The exposure to hi-WY reduced the quantity of plasma FC, C-HDL, and NEFA. Microsomal triglyceride transfer protein and apolipoprotein E mRNA expression were higher in hi-WY, and indicated an increased formation of VLDL particles and energy mobilization from liver. It is speculated that energy depletion by PPARα agonists may contribute to a higher susceptibility to environmental stressors. PMID:25974001

  18. A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

    PubMed

    Howles, Paul A; Gebbie, Leigh K; Collings, David A; Varsani, Arvind; Broad, Ronan C; Ohms, Stephen; Birch, Rosemary J; Cork, Ann H; Arioli, Tony; Williamson, Richard E

    2016-05-01

    The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins. PMID:27008640

  19. The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501

    PubMed Central

    Zhan, Yuhua; Yan, Yongliang; Deng, Zhiping; Chen, Ming; Lu, Wei; Lu, Chao; Shang, Liguo; Yang, Zhimin; Zhang, Wei; Wang, Wei; Li, Yun; Ke, Qi; Lu, Jiasi; Xu, Yuquan; Zhang, Liwen; Xie, Zhihong; Cheng, Qi; Elmerich, Claudine; Lin, Min

    2016-01-01

    Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks. PMID:27407147

  20. The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501.

    PubMed

    Zhan, Yuhua; Yan, Yongliang; Deng, Zhiping; Chen, Ming; Lu, Wei; Lu, Chao; Shang, Liguo; Yang, Zhimin; Zhang, Wei; Wang, Wei; Li, Yun; Ke, Qi; Lu, Jiasi; Xu, Yuquan; Zhang, Liwen; Xie, Zhihong; Cheng, Qi; Elmerich, Claudine; Lin, Min

    2016-07-26

    Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks. PMID:27407147

  1. Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

    PubMed Central

    2010-01-01

    Background The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. Results IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p < 0.001), bronchioles (0.294 ± 0.139 vs. 0.646 ± 0.172, p < 0.001) and in the septal interstitium (0.027 ± 0.014 vs. 0.067 ± 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002) and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001). Conclusions Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment. PMID:20047687

  2. Genome-wide analysis of differential mRNA expression of Amsacta moorei entomopoxvirus, mediated by the gene encoding a viral protein kinase (AMV197).

    PubMed

    Muratoglu, Hacer; Nalcacioglu, Remziye; Arif, Basil M; Demirbag, Zihni

    2016-04-01

    Insect-born entomopoxviruses (Fam: Poxviridae) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus. Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha (ef1a) gene) was downregulated in the absence of amv197. Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process. PMID:26820433

  3. VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level

    PubMed Central

    Arcondéguy, Tania; Lacazette, Eric; Millevoi, Stefania; Prats, Hervé; Touriol, Christian

    2013-01-01

    Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3′-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3′ untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation. PMID:23851566

  4. Imaging Real-Time Gene Expression in Living Systems with Single-Transcript Resolution: Image Analysis of Single mRNA Transcripts

    PubMed Central

    Wells, Amber L.; Condeelis, John S.; Singer, Robert H.; Zenklusen, Daniel

    2016-01-01

    This protocol describes a method for establishing a green fluorescent protein (GFP) calibration curve using dilutions of recombinant GFP and blue fluorescent beads. The total fluorescence intensity (TFI) per mRNA molecule is first calculated by imaging serial dilutions of purified enhanced GFP (eGFP) to determine the TFI within a specific volume. A calibration curve of fluorescence intensity in a given voxel per molecule of GFP is then used to determine the number of GFP molecules in the sample of formaldehyde fixed cells to be imaged. This is followed by a method for detection of single molecules in formaldehyde-fixed and live cells. These cells have been cotransfected with mRNA reporter and MCP-xFP plasmids, where MCP-xFP refers to a fluorescent protein fused to the MS2 capsid protein. It is important to collect micrographs and establish the calibration curve on the same day that the cells are imaged, using the same equipment configuration, camera settings, and image acquisition parameters. Single-molecule measurements using GFP are performed as in Femino et al. (1998, 2003) and Fusco et al. (2003). PMID:21356979

  5. Nucleotide sequence of the gene coding for yeast cytoplasmic aspartyl-tRNA synthetase (APS); mapping of the 5' and 3' termini of AspRS mRNA.

    PubMed Central

    Sellami, M; Fasiolo, F; Dirheimer, G; Ebel, J P; Gangloff, J

    1986-01-01

    A 3.8 Kb DNA fragment, which contains the structural gene of aspartyl-tRNA synthetase (AspRS) and its flanking regions, has been fully sequenced by the combined M13/dideoxy chain terminator method. From the single open reading frame of correct length (1671 bp) we deduced an amino acid sequence consistent with that of several peptides of AspRS. No significant internal sequence repeats were observed in the primary structure of the protein. The AspRS gene (APS) has a codon usage pattern typical of non abundant proteins. S1 nuclease analysis of APS mRNA showed a major start 17 bases downstream from a "TATA box" and stops near an RNA polymerase terminator sequence. Images PMID:3513127

  6. Identification of novel mRNA transcripts of the nm23-M1 gene that are modulated during mouse embryo development and are differently expressed in adult murine tissues.

    PubMed

    Gervasi, F; Capozza, F; Bruno, T; Fanciulli, M; Lombardi, D

    1998-12-01

    The nm23-M1, a putative metastasis-suppressor gene, and its homologs are involved in development and differentiation. We have shown previously that in vitro neuronal cell proliferation and differentiation can be modulated by nm23-M1 expression levels. In the present study, by the yeast two-hybrid system, we have shown that, at the onset of mouse tissue differentiation, the Nm23-M1 protein forms either homodimers, or heterodimers with Nm23-M2. Furthermore, we have isolated two cDNA variants of the nm23-M1 gene in the 3'-untranslated region (UTR). The two variants related to novel mRNA transcripts that are modulated in mouse embryo and are differently expressed in adult murine tissues. PMID:9881672

  7. mRNA imprinting

    PubMed Central

    2011-01-01

    Following its synthesis in the nucleus, mRNA undergoes various stages that are critical for the proper synthesis, localization and possibly functionality of its encoded protein. Recently, we have shown that two RNA polymerase II (Pol II) subunits, Rpb4p and Rpb7p, associate with the nascent transcript co-transcriptionally. This “mRNA imprinting” lasts throughout the mRNA lifetime and is required for proper regulation of all major stages that the mRNA undergoes. Other possible cases of co-transcriptional imprinting are discussed. Since mRNAs can be transported from the synthesizing cell to other cells, we propose that mRNA imprinting can also affect the phenotype of the recipient cells. This can be viewed as “mRNA-based epigenetics.” PMID:21686103

  8. Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

    PubMed Central

    Bhatia, Vikram; Makker, Annu; Tewari, Shikha; Yadu, Alka; Shilpi, Priyanka; Kumar, Sandeep; Agarwal, S. P.; Goel, Sudhir K.

    2014-01-01

    Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC. PMID:24991542

  9. The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri.

    PubMed

    Ni, Duojiao; Song, Linsheng; Gao, Qiang; Wu, Longtao; Yu, Yundong; Zhao, Jianmin; Qiu, Limei; Zhang, Huan; Shi, Fangfang

    2007-11-01

    Cu, Zn superoxide dismutases (SODs) are metalloenzymes that represent one important line of defence against reactive oxygen species (ROS). A cytoplasmic Cu, Zn SOD cDNA sequence was cloned from scallop Chlamys farreri by the homology-based cloning technique. The full-length cDNA of scallop cytoplasmic Cu, Zn SOD (designated CfSOD) was 1022 bp with a 459 bp open reading frame encoding a polypeptide of 153 amino acids. The predicted amino acid sequence of CfSOD shared high identity with cytoplasmic Cu, Zn SOD in molluscs, insects, mammals and other animals, such as cytoplasmic Cu, Zn SOD in oyster Crassostrea gigas (CAD42722), mosquito Aedes aegypti (ABF18094), and cow Bos taurus (XP_584414). A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with Listonella anguillarum, Micrococcus luteus and Candida lipolytica respectively. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD dropped in the first 8-16 h and then recovered after challenge with L. anguillarum and M. luteus, but no change was induced by the C. lipolytica challenge. The results indicated that CfSOD was a constitutive and inducible acute-phase protein, and could play an important role in the immune responses against L. anguillarum and M. luteus infection. PMID:17574438

  10. Increased MET Gene Copy Number but Not mRNA Level Predicts Postoperative Recurrence in Patients with Non–Small Cell Lung Cancer1

    PubMed Central

    Kowalczuk, Oksana; Kozlowski, Miroslaw; Niklinska, Wiesława; Kisluk, Joanna; Niklinska, Barbara Joanna; Niklinski, Jacek

    2014-01-01

    The aim of the present study was to investigate the relationship of MET copy number (CN) and MET mRNA expression to other molecular alterations, clinicopathologic characteristics, and survival of patients with resected non–small cell lung cancer. One hundred fifty-one paired surgical samples of tumor and tumor-distant normal lung tissues were analyzed by comparative quantitative polymerase chain reaction (PCR) methods with commercially available assays and the CopyCaller software v. 1.0 for post-PCR data processing (downloadable from www.appliedbiosystems.com). MET copy gain (set as more than 3.0 copies per cell) was found in 18.5% of the samples and occurred more frequently in the adenocarcinomas (ADCs) with an increased epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) CN (P = .001 and .030 for EGFR and HER2, respectively) and in the ADCs with EGFR activating mutations (P = .051) but did not correlate with KRAS dosage or mutational status. MET mRNA level was 1.76-fold higher [95% confidence interval (CI), 1.29-2.40] in the tumor compared to unaffected lung tissue and associated significantly with MET CN (beta coefficient, 1.51; 95% CI, 1.22-1.87; P < .001). In the multivariable analysis, patients diagnosed with ADC with increased MET CN had a significantly higher risk of disease recurrence (hazard ratio, 1.76; 95% CI, 1.20-2.57; P = .004). An increased MET CN in combination with histologic type appears to be a prognostic factor in patients with ADC after a curative surgery. PMID:25389455